Familial FTDP-17 Missense Mutations Inhibit Microtubule Assembly-promoting Activity of Tau by Increasing Phosphorylation at Ser202 in Vitro

In Alzheimer disease (AD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and other tauopathies, tau accumulates and forms paired helical filaments (PHFs) in the brain. Tau isolated from PHFs is phosphorylated at a number of sites, migrates as ∼60-, 64-, and 68-kDa bands on SDS-gel, and does not promote microtubule assembly. Upon dephosphorylation, the PHF-tau migrates as ∼50–60-kDa bands on SDS-gels in a manner similar to tau that is isolated from normal brain and promotes microtubule assembly. The site(s) that inhibits microtubule assembly-promoting activity when phosphorylated in the diseased brain is not known. In this study, when tau was phosphorylated by Cdk5 in vitro, its mobility shifted from ∼60-kDa bands to ∼64- and 68-kDa bands in a time-dependent manner. This mobility shift correlated with phosphorylation at Ser²⁰², and Ser²⁰² phosphorylation inhibited tau microtubule-assembly promoting activity. When several tau point mutants were analyzed, G272V, P301L, V337M, and R406W mutations associated with FTDP-17, but not nonspecific mutations S214A and S262A, promoted Ser²⁰² phosphorylation and mobility shift to a ∼68-kDa band. Furthermore, Ser²⁰² phosphorylation inhibited the microtubule assembly-promoting activity of FTDP-17 mutants more than of WT. Our data indicate that FTDP-17 missense mutations, by promoting phosphorylation at Ser²⁰², inhibit the microtubule assembly-promoting activity of tau in vitro, suggesting that Ser²⁰² phosphorylation plays a major role in the development of NFT pathology in AD and related tauopathies.Neurofibrillary tangles (NFTs)⁴ and senile plaques are the two characteristic neuropathological lesions found in the brains of patients suffering from Alzheimer disease (AD). The major fibrous component of NFTs are paired helical filaments (PHFs) (for reviews see Refs. 1–3). Initially, PHFs were found to be composed of a protein component referred to as “A68” (4). Biochemical analysis reveled that A68 is identical to the microtubule-associated protein, tau (4, 5). Some characteristic features of tau isolated from PHFs (PHF-tau) are that it is abnormally hyperphosphorylated (phosphorylated on more sites than the normal brain tau), does not bind to microtubules, and does not promote microtubule assembly in vitro. Upon dephosphorylation, PHF-tau regains its ability to bind to and promote microtubule assembly (6, 7). Tau hyperphosphorylation is suggested to cause microtubule instability and PHF formation, leading to NFT pathology in the brain (1–3).PHF-tau is phosphorylated on at least 21 proline-directed and non-proline-directed sites (8, 9). The individual contribution of these sites in converting tau to PHFs is not entirely clear. However, some sites are only partially phosphorylated in PHFs (8), whereas phosphorylation on specific sites inhibits the microtubule assembly-promoting activity of tau (6, 10). These observations suggest that phosphorylation on a few sites may be responsible and sufficient for causing tau dysfunction in AD.Tau purified from the human brain migrates as ∼50–60-kDa bands on SDS-gel due to the presence of six isoforms that are phosphorylated to different extents (2). PHF-tau isolated from AD brain, on the other hand, displays ∼60-, 64-, and 68 kDa-bands on an SDS-gel (4, 5, 11). Studies have shown that ∼64- and 68-kDa tau bands (the authors have described the ∼68-kDa tau band as an ∼69-kDa band in these studies) are present only in brain areas affected by NFT degeneration (12, 13). Their amount is correlated with the NFT densities at the affected brain regions. Moreover, the increase in the amount of ∼64- and 68-kDa band tau in the brain correlated with a decline in the intellectual status of the patient. The ∼64- and 68-kDa tau bands were suggested to be the pathological marker of AD (12, 13). Biochemical analyses determined that ∼64- and 68-kDa bands are hyperphosphorylated tau, which upon dephosphorylation, migrated as normal tau on SDS-gel (4, 5, 11). Tau sites involved in the tau mobility shift to ∼64- and 68-kDa bands were suggested to have a role in AD pathology (12, 13). It is not known whether phosphorylation at all 21 PHF-sites is required for the tau mobility shift in AD. However, in vitro the tau mobility shift on SDS-gel is sensitive to phosphorylation only on some sites (6, 14). It is therefore possible that in the AD brain, phosphorylation on some sites also causes a tau mobility shift. Identification of such sites will significantly enhance our knowledge of how NFT pathology develops in the brain.PHFs are also the major component of NFTs found in the brains of patients suffering from a group of neurodegenerative disorders collectively called tauopathies (2, 11). These disorders include frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17), corticobasal degeneration, progressive supranuclear palsy, and Pick disease. Each PHF-tau isolated from autopsied brains of patients suffering from various tauopathies is hyperphosphorylated, displays ∼60-, 64-, and 68-kDa bands on SDS-gel, and is incapable of binding to microtubules. Upon dephosphorylation, the above referenced PHF-tau migrates as a normal tau on SDS-gel, binds to microtubules, and promotes microtubule assembly (2, 11). These observations suggest that the mechanisms of NFT pathology in various tauopathies may be similar and the phosphorylation-dependent mobility shift of tau on SDS-gel may be an indicator of the disease. The tau gene is mutated in familial FTDP-17, and these mutations accelerate NFT pathology in the brain (15–18). Understanding how FTDP-17 mutations promote tau phosphorylation can provide a better understanding of how NFT pathology develops in AD and various tauopathies. However, when expressed in CHO cells, G272V, R406W, V337M, and P301L tau mutations reduce tau phosphorylation (19, 20). In COS cells, although G272V, P301L, and V337M mutations do not show any significant affect, the R406W mutation caused a reduction in tau phosphorylation (21, 22). When expressed in SH-SY5Y cells subsequently differentiated into neurons, the R406W, P301L, and V337M mutations reduce tau phosphorylation (23). In contrast, in hippocampal neurons, R406W increases tau phosphorylation (24). When phosphorylated by recombinant GSK3β in vitro, the P301L and V337M mutations do not have any effect, and the R406W mutation inhibits phosphorylation (25). However, when incubated with rat brain extract, all of the G272V, P301L, V337M, and R406W mutations stimulate tau phosphorylation (26). The mechanism by which FTDP-17 mutations promote tau phosphorylation leading to development of NFT pathology has remained unclear.Cyclin-dependent protein kinase 5 (Cdk5) is one of the major kinases that phosphorylates tau in the brain (27, 28). In this study, to determine how FTDP-17 missense mutations affect tau phosphorylation, we phosphorylated four FTDP-17 tau mutants (G272V, P301L, V337M, and R406W) by Cdk5. We have found that phosphorylation of tau by Cdk5 causes a tau mobility shift to ∼64- and 68 kDa-bands. Although the mobility shift to a ∼64-kDa band is achieved by phosphorylation at Ser^396/404 or Ser²⁰², the mobility shift to a 68-kDa band occurs only in response to phosphorylation at Ser²⁰². We show that in vitro, FTDP-17 missense mutations, by promoting phosphorylation at Ser²⁰², enhance the mobility shift to ∼64- and 68-kDa bands and inhibit the microtubule assembly-promoting activity of tau. Our data suggest that Ser²⁰² phosphorylation is the major event leading to NFT pathology in AD and related tauopathies.     

Mammalian Target of Rapamycin Complex 1 (mTORC1) Activity Is Associated with Phosphorylation of Raptor by mTOR

mTORC1 contains multiple proteins and plays a central role in cell growth and metabolism. Raptor (regulatory-associated protein of mammalian target of rapamycin (mTOR)), a constitutively binding protein of mTORC1, is essential for mTORC1 activity and critical for the regulation of mTORC1 activity in response to insulin signaling and nutrient and energy sufficiency. Herein we demonstrate that mTOR phosphorylates raptor in vitro and in vivo. The phosphorylated residues were identified by using phosphopeptide mapping and mutagenesis. The phosphorylation of raptor is stimulated by insulin and inhibited by rapamycin. Importantly, the site-directed mutation of raptor at one phosphorylation site, Ser⁸⁶³, reduced mTORC1 activity both in vitro and in vivo. Moreover, the Ser⁸⁶³ mutant prevented small GTP-binding protein Rheb from enhancing the phosphorylation of S6 kinase (S6K) in cells. Therefore, our findings indicate that mTOR-mediated raptor phosphorylation plays an important role on activation of mTORC1.Mammalian target of rapamycin (mTOR)² has been shown to function as a critical controller in cellular growth, survival, metabolism, and development (1). mTOR, a highly conserved Ser-Thr phosphatidylinositol 3-kinase-related protein kinase, structurally forms two distinct complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), each of which catalyzes the phosphorylation of different substrates (1). The best characterized substrates for mTORC1 are eIF4E-binding protein (4E-BP, also known as PHAS) and p70 S6 kinase (S6K) (1), whereas mTORC2 phosphorylates the hydrophobic and turn motifs of protein kinase B (Akt/protein kinase B) (2) and protein kinase C (3, 4). mTORC1 constitutively consists of mTOR, raptor, and mLst8/GβL (1), whereas the proline-rich Akt substrate of 40 kDa (PRAS40) is a regulatory component of mTORC1 that disassociates after growth factor stimulation (5, 6). Raptor is essential for mTORC1 activity by providing a substrate binding function (7) but also plays a regulatory role on mTORC1 with stimuli of growth factors and nutrients (8). In response to insulin, raptor binding to substrates is elevated through the release of the competitive inhibitor PRAS40 from mTORC1 (9, 10) because PRAS40 and the substrates of mTORC1 (4E-BP and S6K) appear to bind raptor through a consensus sequence, the TOR signaling (TOS) motif (10–14). In response to amino acid sufficiency, raptor directly interacts with a heterodimer of Rag GTPases and promotes mTORC1 localization to the Rheb-containing vesicular compartment (15).mTORC1 integrates signaling pathways from growth factors, nutrients, energy, and stress, all of which generally converge on the tuberous sclerosis complex (TSC1-TSC2) through the phosphorylation of TSC2 (1). Growth factors inhibit the GTPase-activating protein activity of TSC2 toward the small GTPase Rheb via the PI3K/Akt pathway (16, 17), whereas energy depletion activates TSC2 GTPase-activating protein activity by stimulating AMP-activated protein kinase (AMPK) (18). Rheb binds directly to mTOR, albeit with very low affinity (19), and upon charging with GTP, Rheb functions as an mTORC1 activator (6). mTORC1 complexes isolated from growth factor-stimulated cells show increased kinase activity yet do not contain detectable levels of associated Rheb. Therefore, how Rheb-GTP binding to mTOR leads to an increase in mTORC1 activity toward substrates, and what the role of raptor is in this activation is currently unknown. More recently, the AMPK and p90 ribosomal S6 kinase (RSK) have been reported to directly phosphorylate raptor and regulate mTORC1 activity. The phosphorylation of raptor directly by AMPK reduced mTORC1 activity, suggesting an alternative regulation mechanism independent of TSC2 in response to energy supply (20). RSK-mediated raptor phosphorylation enhances mTORC1 activity and provides a mechanism whereby stress may activate mTORC1 independent of the PI3K/Akt pathway (21). Therefore, the phosphorylation status of raptor can be critical for the regulation of mTORC1 activity.In this study, we investigated phosphorylation sites in raptor catalyzed by mTOR. Using two-dimensional phosphopeptide mapping, we found that Ser⁸⁶³ and Ser⁸⁵⁹ in raptor were phosphorylated by mTOR both in vivo and in vitro. mTORC1 activity in vitro and in vivo is associated with the phosphorylation of Ser⁸⁶³ in raptor.     

Role of JNK1-dependent Bcl-2 Phosphorylation in Ceramide-induced Macroautophagy

Macroautophagy is a vacuolar lysosomal catabolic pathway that is stimulated during periods of nutrient starvation to preserve cell integrity. Ceramide is a bioactive sphingolipid associated with a large range of cell processes. Here we show that short-chain ceramides (C₂-ceramide and C₆-ceramide) and stimulation of the de novo ceramide synthesis by tamoxifen induce the dissociation of the complex formed between the autophagy protein Beclin 1 and the anti-apoptotic protein Bcl-2. This dissociation is required for macroautophagy to be induced either in response to ceramide or to starvation. Three potential phosphorylation sites, Thr⁶⁹, Ser⁷⁰, and Ser⁸⁷, located in the non-structural N-terminal loop of Bcl-2, play major roles in the dissociation of Bcl-2 from Beclin 1. We further show that activation of c-Jun N-terminal protein kinase 1 by ceramide is required both to phosphorylate Bcl-2 and to stimulate macroautophagy. These findings reveal a new aspect of sphingolipid signaling in up-regulating a major cell process involved in cell adaptation to stress.Macroautophagy (referred to below as “autophagy”) is a vacuolar, lysosomal degradation pathway for cytoplasmic constituents that is conserved in eukaryotic cells (1–3). Autophagy is initiated by the formation of a multimembrane-bound autophagosome that engulfs cytoplasmic proteins and organelles. The last stage in the process results in fusion with the lysosomal compartments, where the autophagic cargo undergoes degradation. Basal autophagy is important in controlling the quality of the cytoplasm by removing damaged organelles and protein aggregates. Inhibition of basal autophagy in the brain is deleterious, and leads to neurodegeneration in mouse models (4, 5). Stimulation of autophagy during periods of nutrient starvation is a physiological response present at birth and has been shown to provide energy in various tissues of newborn pups (6). In cultured cells, starvation-induced autophagy is an autonomous cell survival mechanism, which provides nutrients to maintain a metabolic rate and level of ATP compatible with cell survival (7). In addition, starvation-induced autophagy blocks the induction of apoptosis (8). In other contexts, such as drug treatment and a hypoxic environment, autophagy has also been shown to be cytoprotective in cancer cells (9, 10). However, autophagy is also part of cell death pathways in certain situations (11). Autophagy can be a player in apoptosis-independent type-2 cell death (type-1 cell death is apoptosis), also known as autophagic cell death. This situation has been shown to occur when the apoptotic machinery is crippled in mammalian cells (12, 13). Autophagy can also be part of the apoptotic program, for instance in tumor necrosis factor-α-induced cell death when NF-κB is inhibited (14), or in human immunodeficiency virus envelope-mediated cell death in bystander naive CD4 T cells (15). Moreover autophagy has recently been shown to be required for the externalization of phosphatidylserine, the eat-me signal for phagocytic cells, at the surface of apoptotic cells (16).The complex relationship between autophagy and apoptosis reflects the intertwined regulation of these processes (17, 18). Many signaling pathways involved in the regulation of autophagy also regulate apoptosis. This intertwining has recently been shown to occur at the level of the molecular machinery of autophagy. In fact the anti-apoptotic protein Bcl-2 has been shown to inhibit starvation-induced autophagy by interacting with the autophagy protein Beclin 1 (19). Beclin 1 is one of the Atg proteins conserved from yeast to humans (it is the mammalian orthologue of yeast Atg6) and is involved in autophagosome formation (20). Beclin 1 is a platform protein that interacts with several different partners, including hVps34 (class III phosphatidylinositol 3-kinase), which is responsible for the synthesis of phosphatidylinositol 3-phosphate. The production of this lipid is important for events associated with the nucleation of the isolation membrane before it elongates and closes to form autophagosomes in response to other Atg proteins, including the Atg12 and LC3² (microtubule-associated protein light chain 3 is the mammalian orthologue of the yeast Atg8) ubiquitin-like conjugation systems (3, 21). Various partners associated with the Beclin 1 complex modulate the activity of hVps34. For instance, Bcl-2 inhibits the activity of this enzyme, whereas UVRAG, Ambra-1, and Bif-1 all up-regulate it (22, 23).In view of the intertwining between autophagy and apoptosis, it is noteworthy that Beclin 1 belongs to the BH3-only family of proteins (24–26). However, and unlike most of the proteins in this family, Beclin 1 is not able to trigger apoptosis when its expression is forced in cells (27). A BH3-mimetic drug, ABT-737, is able to dissociate the Beclin 1-Bcl-2 complex, and to trigger autophagy by mirroring the effect of starvation (25).The sphingolipids constitute a family of bioactive lipids (28–32) of which several members, such as ceramide and sphingosine 1-phosphate, are signaling molecules. These molecules constitute a “sphingolipid rheostat” that determines the fate of the cell, because in many settings ceramide is pro-apoptotic and sphingosine 1-phosphate mitigates this apoptotic effect (31, 32). However, ceramide is also engaged in a wide variety of other cell processes, such as the formation of exosomes (33), differentiation, cell proliferation, and senescence (34). Recently we showed that both ceramide and sphingosine 1-phosphate are able to stimulate autophagy (35, 36). It has also been shown that ceramide triggers autophagy in a large panel of mammalian cells (37–39). However, elucidation of the mechanism by which ceramide stimulates autophagy is still in its infancy. We have previously demonstrated that ceramide induces autophagy in breast and colon cancer cells by inhibiting the Class I phosphatidylinositol 3-phosphate/mTOR signaling pathway, which plays a central role in inhibiting autophagy (36). Inhibition of mTOR is another hallmark of starvation-induced autophagy (17). This finding led us to investigate the effect of ceramide on the Beclin 1-Bcl-2 complex. The results presented here show that ceramide is more potent than starvation in dissociating the Beclin 1-Bcl-2 complex (see Ref. 40). This dissociation is dependent on three phosphorylation sites (Thr⁶⁹, Ser⁷⁰, and Ser⁸⁷) located in a non-structural loop of Bcl-2. Ceramide induces the c-Jun N-terminal kinase 1-dependent phosphorylation of Bcl-2. Expression of a dominant negative form of JNK1 blocks Bcl-2 phosphorylation, and thus the induction of autophagy by ceramide. These findings help to explain how autophagy is regulated by a major lipid second messenger.     

Protein Kinase D Mediates Mitogenic Signaling by Gq-coupled Receptors through Protein Kinase C-independent Regulation of Activation Loop Ser744 and Ser748 Phosphorylation

Rapid protein kinase D (PKD) activation and phosphorylation via protein kinase C (PKC) have been extensively documented in many cell types cells stimulated by multiple stimuli. In contrast, little is known about the role and mechanism(s) of a recently identified sustained phase of PKD activation in response to G protein-coupled receptor agonists. To elucidate the role of biphasic PKD activation, we used Swiss 3T3 cells because PKD expression in these cells potently enhanced duration of ERK activation and DNA synthesis in response to G_q-coupled receptor agonists. Cell treatment with the preferential PKC inhibitors GF109203X or Gö6983 profoundly inhibited PKD activation induced by bombesin stimulation for <15 min but did not prevent PKD catalytic activation induced by bombesin stimulation for longer times (>60 min). The existence of sequential PKC-dependent and PKC-independent PKD activation was demonstrated in 3T3 cells stimulated with various concentrations of bombesin (0.3–10 nm) or with vasopressin, a different G_q-coupled receptor agonist. To gain insight into the mechanisms involved, we determined the phosphorylation state of the activation loop residues Ser⁷⁴⁴ and Ser⁷⁴⁸. Transphosphorylation targeted Ser⁷⁴⁴, whereas autophosphorylation was the predominant mechanism for Ser⁷⁴⁸ in cells stimulated with G_q-coupled receptor agonists. We next determined which phase of PKD activation is responsible for promoting enhanced ERK activation and DNA synthesis in response to G_q-coupled receptor agonists. We show, for the first time, that the PKC-independent phase of PKD activation mediates prolonged ERK signaling and progression to DNA synthesis in response to bombesin or vasopressin through a pathway that requires epidermal growth factor receptor-tyrosine kinase activity. Thus, our results identify a novel mechanism of G_q-coupled receptor-induced mitogenesis mediated by sustained PKD activation through a PKC-independent pathway.The understanding of the mechanisms that control cell proliferation requires the identification of the molecular pathways that govern the transition of quiescent cells into the S phase of the cell cycle. In this context the activation and phosphorylation of protein kinase D (PKD),⁴ the founding member of a new protein kinase family within the Ca²⁺/calmodulin-dependent protein kinase (CAMK) group and separate from the previously identified PKCs (for review, see Ref. 1), are attracting intense attention. In unstimulated cells, PKD is in a state of low catalytic (kinase) activity maintained by autoinhibition mediated by the N-terminal domain, a region containing a repeat of cysteinerich zinc finger-like motifs and a pleckstrin homology (PH) domain (1–4). Physiological activation of PKD within cells occurs via a phosphorylation-dependent mechanism first identified in our laboratory (5–7). In response to cellular stimuli (1), including phorbol esters, growth factors (e.g. PDGF), and G protein-coupled receptor (GPCR) agonists (6, 8–16) that signal through G_q, G₁₂, G_i, and Rho (11, 15–19), PKD is converted into a form with high catalytic activity, as shown by in vitro kinase assays performed in the absence of lipid co-activators (5, 20).During these studies multiple lines of evidence indicated that PKC activity is necessary for rapid PKD activation within intact cells. For example, rapid PKD activation was selectively and potently blocked by cell treatment with preferential PKC inhibitors (e.g. GF109203X or Gö6983) that do not directly inhibit PKD catalytic activity (5, 20), implying that PKD activation in intact cells is mediated directly or indirectly through PKCs. Many reports demonstrated the operation of a rapid PKC/PKD signaling cascade induced by multiple GPCR agonists and other receptor ligands in a range of cell types (for review, see Ref. 1). Our previous studies identified Ser⁷⁴⁴ and Ser⁷⁴⁸ in the PKD activation loop (also referred as activation segment or T-loop) as phosphorylation sites critical for PKC-mediated PKD activation (1, 4, 7, 17, 21). Collectively, these findings demonstrated the existence of a rapidly activated PKC-PKD protein kinase cascade(s). In a recent study we found that the rapid PKC-dependent PKD activation was followed by a late, PKC-independent phase of catalytic activation and phosphorylation induced by stimulation of the bombesin G_q-coupled receptor ectopically expressed in COS-7 cells (22). This study raised the possibility that PKD mediates rapid biological responses downstream of PKCs, whereas, in striking contrast, PKD could mediate long term responses through PKC-independent pathways. Despite its potential importance for defining the role of PKC and PKD in signal transduction, this hypothesis has not been tested in any cell type.Accumulating evidence demonstrates that PKD plays an important role in several cellular processes and activities, including signal transduction (14, 23–25), chromatin organization (26), Golgi function (27, 28), gene expression (29–31), immune regulation (26), and cell survival, adhesion, motility, differentiation, DNA synthesis, and proliferation (for review, see Ref. 1). In Swiss 3T3 fibroblasts, a cell line used extensively as a model system to elucidate mechanisms of mitogenic signaling (32–34), PKD expression potently enhances ERK activation, DNA synthesis, and cell proliferation induced by G_q-coupled receptor agonists (8, 14). Here, we used this model system to elucidate the role and mechanism(s) of biphasic PKD activation. First, we show that the G_q-coupled receptor agonists bombesin and vasopressin, in contrast to phorbol esters, specifically induce PKD activation through early PKC-dependent and late PKC-independent mechanisms in Swiss 3T3 cells. Subsequently, we demonstrate for the first time that the PKC-independent phase of PKD activation is responsible for promoting ERK signaling and progression to DNA synthesis through an epidermal growth factor receptor (EGFR)-dependent pathway. Thus, our results identify a novel mechanism of G_q-coupled receptor-induced mitogenesis mediated by sustained PKD activation through a PKC-independent pathway.     

The Death Effector Domain-containing DEDD Supports S6K1 Activity via Preventing Cdk1-dependent Inhibitory Phosphorylation

Cell cycle regulation and biochemical responses upon nutrients and growth factors are the major regulatory mechanisms for cell sizing in mammals. Recently, we identified that the death effector domain-containing DEDD impedes mitotic progression by inhibiting Cdk1 (cyclin-dependent kinase 1) and thus maintains an increase of cell size during the mitotic phase. Here we found that DEDD also associates with S6 kinase 1 (S6K1), downstream of phosphatidylinositol 3-kinase, and supports its activity by preventing inhibitory phosphorylation of S6K1 brought about by Cdk1 during the mitotic phase. DEDD^-/- cells showed reduced S6K1 activity, consistently demonstrating decreased levels in activating phosphorylation at the Thr-389 site. In addition, levels of Cdk1-dependent inhibitory phosphorylation at the C terminus of S6K1 were enhanced in DEDD^-/- cells and tissues. Consequently, as in S6K1^-/- mice, the insulin mass within pancreatic islets was reduced in DEDD^-/- mice, resulting in glucose intolerance. These findings suggest a novel cell sizing mechanism achieved by DEDD through the maintenance of S6K1 activity prior to cell division. Our results also suggest that DEDD may harbor important roles in glucose homeostasis and that its deficiency might be involved in the pathogenesis of type 2 diabetes mellitus.Cell size is closely related to specialized cell function and to the specific patterning of tissues in the body. Cell sizing is regulated mainly by two mechanisms: cell cycle control and the biochemical response to nutrients and/or growth factors (1–5). During cell cycle progression, both the G₁ (which is believed to be dominant) and the G₂ periods are important for cells to increase their volume (6–9). In addition, we recently provided evidence that the mitotic period (M phase) also influences cell size, through analysis of DEDD-deficient mice (10, 11). The DEDD molecule was initially described as a member of the death effector domain (DED)²-containing protein family (12). Although the absence of DEDD did not apparently influence progression of apoptosis (10), we found that during mitosis, DEDD is associated with Cdk1-cyclin B1 and that it decreases the kinase activity of Cdk1. This response impedes the Cdk1-dependent mitotic program to shut off synthesis of ribosomal RNA (rRNA) and protein and is consequently useful in gaining sufficient cell growth prior to cell division. Depletion of DEDD consistently results in a shortened mitotic duration and an overall reduction in the amount of cellular rRNA and protein and, furthermore, in cell and body size (10, 11).Of the biochemical responses responsible for cell sizing, the signaling cascade involving phosphatydilinositol 3-kinase (PI3K) and its downstream target of rapamycin (TOR) is most crucial (13–15). In mammals, upon stimulation by growth factors, including insulin, the mammalian TOR (mTOR) cooperates with PI3K-dependent effectors to activate S6K1, thereby phosphorylating the 40 S ribosomal protein S6, and subsequently enhances translation of the 5′-terminal oligopyrimidine sequences that encode components of the translational machinery. This reaction increases the number of ribosomes and the efficacy of protein synthesis, thus critically promoting cell growth (16–18). Therefore, mice deficient for S6K1 (S6K1^-/-) had reduced cell and body size (19–23). This effect also involves S6K1 in maintenance of glucose tolerance. S6K1 significantly supports the size of insulin-producing β cells within pancreatic Langerhans islets (24, 25). Thus, in S6K1^-/- mice, the insulin mass was diminished, which resulted in ineffective secretion of insulin upon glucose administration (21, 23).The activation of S6K1 proceeds through chronological phosphorylation at various residues, toward the crucial phosphorylation of Thr-389, present within the linker domain between the catalytic domain and the carboxyl tail, to obtain maximal enzymatic activity (26). Interestingly, phosphorylation at several Ser/Thr residues within the C-terminal autoinhibitory tail appears to either activate or inhibit S6K1, depending on the cell cycle phase. Shah et al. (27) demonstrated that phosphorylation of those residues (featured by the Thr-421/Ser-424 site) during mitosis pursued by Cdk1 inactivates S6K1 to terminate protein synthesis prior to cell division (28). A recent report by Schmidt et al. (29) demonstrating that phosphorylation of Thr-421/Ser-424 is specifically increased during the G₂/M phase may also support the finding, whereas during the G₁ phase, there is consensus that the phosphorylation at the autoinhibitory domain is requisite for S6K1 activation (26), as also recently demonstrated by Hou et al. (30), where the Cdk5 phosphorylates the Ser-411 site, leading to activation of S6K1. In contrast to such inhibitory regulation of S6K1 during mitosis, however, a recent report by Boyer et al. (31) sharply demonstrated that the activity of S6K1 peaks at mitosis, suggesting that S6K1 may also have some roles during the mitotic phase. If so, how its activity is supported against the inhibitory regulation caused by Cdk1 remains an open question.Hence, the two observations above that both DEDD^-/- and S6K1^-/- situations decrease the efficacy of ribosome and protein synthesis, resulting in smaller cell and body size, and that mitotic Cdk1 has a functional interaction with both S6K1 and DEDD led us here to assess a possible role of DEDD in the context of the functional regulation of S6K1.     

G Protein-coupled Receptor Kinase-mediated Phosphorylation Regulates Post-endocytic Trafficking of the D2 Dopamine Receptor

We investigated the role of G protein-coupled receptor kinase (GRK)-mediated phosphorylation in agonist-induced desensitization, arrestin association, endocytosis, and intracellular trafficking of the D₂ dopamine receptor (DAR). Agonist activation of D₂ DARs results in rapid and sustained receptor phosphorylation that is solely mediated by GRKs. A survey of GRKs revealed that only GRK2 or GRK3 promotes D₂ DAR phosphorylation. Mutational analyses resulted in the identification of eight serine/threonine residues within the third cytoplasmic loop of the receptor that are phosphorylated by GRK2/3. Simultaneous mutation of these eight residues results in a receptor construct, GRK(-), that is completely devoid of agonist-promoted GRK-mediated receptor phosphorylation. We found that both wild-type (WT) and GRK(-) receptors underwent a similar degree of agonist-induced desensitization as assessed using [³⁵S]GTPγS binding assays. Similarly, both receptor constructs internalized to the same extent in response to agonist treatment. Furthermore, using bioluminescence resonance energy transfer assays to directly assess receptor association with arrestin3, we found no differences between the WT and GRK(-) receptors. Thus, phosphorylation is not required for arrestin-receptor association or agonist-induced desensitization or internalization. In contrast, when we examined recycling of the D₂ DARs to the cell surface, subsequent to agonist-induced endocytosis, the GRK(-) construct exhibited less recycling in comparison with the WT receptor. This impairment appears to be due to a greater propensity of the GRK(-) receptors to down-regulate once internalized. In contrast, if the receptor is highly phosphorylated, then receptor recycling is promoted. These results reveal a novel role for GRK-mediated phosphorylation in regulating the post-endocytic trafficking of a G protein-coupled receptor.Dopamine receptors (DARs)³ are members of the GPCR superfamily and consist of five structurally distinct subtypes (1, 2). These can be divided into two subfamilies on the basis of their structure and pharmacological and transductional properties (3). The “D₁-like” subfamily includes the D₁ and D₅ receptors, which couple to the heterotrimeric G proteins G_S or G_OLF to stimulate adenylyl cyclase activity and raise intracellular levels of cAMP. The D₂-like subfamily includes the D₂, D₃, and D₄ receptors, which couple to inhibitory G_i/o proteins to reduce adenylyl cyclase activity as well as modulate voltage-gated K⁺ or Ca²⁺ channels. Within the central nervous system, these receptors modulate movement, learning and memory, reward and addiction, cognition, and certain neurendocrine functions. As with other GPCRs, the DARs are subject to a wide variety of regulatory mechanisms, which can either positively or negatively modulate their expression and functional activity (4).Upon agonist activation, most GPCRs undergo desensitization, a homeostatic process that results in a waning of receptor response despite continued agonist stimulation (5, 6). Desensitization is believed to involve the phosphorylation of receptors by either G protein-coupled receptor kinases (GRKs) and/or second messenger-activated kinases such as PKA or PKC. Homologous forms of desensitization involve only agonist-activated receptors and appear to be primarily mediated by GRKs. In many cases, GRK-mediated phosphorylation has been shown to decrease receptor/G protein interactions and also initiate arrestin binding, which further promotes endocytosis of the receptor through clathrin-coated pits (7–9). Once internalized, GPCRs can engage additional signaling pathways (10), be sorted for recycling to the plasma membrane, or targeted for degradation (7–9). Among the DARs, the D₂ receptor is arguably one of the most validated drug targets in neurology and psychiatry. For instance, all receptor-based anti-parkinsonian drugs work via stimulating the D₂ DAR (11), whereas all Food and Drug Administration-approved antipsychotic agents are antagonists of this receptor subtype (12, 13). The D₂ DAR is also therapeutically targeted in other disorders such as restless legs syndrome, tardive dyskinesia, Tourette syndrome, and hyperprolactinemia. As such, more knowledge concerning the regulation of the D₂ DAR could be helpful in improving current therapies or devising new treatment strategies.In comparison with other GPCRs, however, detailed mechanistic information concerning regulation of the D₂ DAR is mostly lacking, although some progress has recently been made. For instance, we (14) and others (15) have found that PKC-mediated phosphorylation can regulate both D₂ receptor desensitization and trafficking. In our PKC study, we mapped out all of the PKC phosphorylation sites within the third intracellular loop (IC3) of the receptor, and we determined the existence of two PKC phosphorylation domains. Both of these domains were found to regulate receptor sequestration, whereas only one domain regulated functional uncoupling in response to PKC activation (14). In response to agonist activation, the D₂ DAR has also been shown to undergo functional desensitization (4), although this has not been intensively investigated. More thoroughly examined is the observation that agonist stimulation of the D₂ DAR promotes its sequestration from the cell surface into vesicular compartments that appear distinct from those harboring internalized D₁ DARs or β-adrenergic receptors (16–21). In addition to uncertainty over the endocytic pathway involved, controversy also exists as to whether or not D₂ DAR internalization is dynamin-dependent and whether the internalized receptors can partially or completely recycle to the cell surface or, alternatively, if they undergo degradation (19, 21–24). The D₂ DAR does appear to undergo GRK-mediated phosphorylation upon agonist activation, which has been suggested to promote arrestin association and receptor sequestration (16, 19, 25), although this process has not been studied in detail and its relationship to functional receptor desensitization is unknown.In this study, we have further characterized GRK-mediated phosphorylation of the D₂ DAR and determined its role in agonist-induced receptor desensitization, internalization, and recycling. Using site-directed mutagenesis, we have mapped out all of the GRK phosphorylation sites within the D₂ DAR and determined that these are distinct from the PKC phosphorylation sites. Using a GRK phosphorylation-null mutant receptor, we found, surprisingly, that GRK-mediated phosphorylation is not actually required for agonist-induced receptor desensitization, arrestin association, or internalization. In contrast, we found that the GRK phosphorylation-null receptor was impaired in its ability to recycle to the cell surface subsequent to internalization and was degraded to a greater extent in comparison with the wild-type receptor. These results suggest that GRK-mediated phosphorylation of the D₂ DAR regulates its intracellular trafficking or sorting once internalized, a novel mechanism for GRK-mediated regulation of GPCR function.     

A Role for Protein Phosphorylation in Cytochrome P450 3A4 Ubiquitin-dependent Proteasomal Degradation

Cytochromes P450 (P450s) incur phosphorylation. Although the precise role of this post-translational modification is unclear, marking P450s for degradation is plausible. Indeed, we have found that after structural inactivation, CYP3A4, the major human liver P450, and its rat orthologs are phosphorylated during their ubiquitin-dependent proteasomal degradation. Peptide mapping coupled with mass spectrometric analyses of CYP3A4 phosphorylated in vitro by protein kinase C (PKC) previously identified two target sites, Thr²⁶⁴ and Ser⁴²⁰. We now document that liver cytosolic kinases additionally target Ser⁴⁷⁸ as a major site. To determine whether such phosphorylation is relevant to in vivo CYP3A4 degradation, wild type and CYP3A4 with single, double, or triple Ala mutations of these residues were heterologously expressed in Saccharomyces cerevisiae pep4Δ strains. We found that relative to CYP3A4wt, its S478A mutant was significantly stabilized in these yeast, and this was greatly to markedly enhanced for its S478A/T264A, S478A/S420A, and S478A/T264A/S420A double and triple mutants. Similar relative S478A/T264A/S420A mutant stabilization was also observed in HEK293T cells. To determine whether phosphorylation enhances CYP3A4 degradation by enhancing its ubiquitination, CYP3A4 ubiquitination was examined in an in vitro UBC7/gp78-reconstituted system with and without cAMP-dependent protein kinase A and PKC, two liver cytosolic kinases involved in CYP3A4 phosphorylation. cAMP-dependent protein kinase A/PKC-mediated phosphorylation of CYP3A4wt but not its S478A/T264A/S420A mutant enhanced its ubiquitination in this system. Together, these findings indicate that phosphorylation of CYP3A4 Ser⁴⁷⁸, Thr²⁶⁴, and Ser⁴²⁰ residues by cytosolic kinases is important both for its ubiquitination and proteasomal degradation and suggest a direct link between P450 phosphorylation, ubiquitination, and degradation.Hepatic cytochromes P450 (P450s)³ are integral endoplasmic reticulum (ER)-anchored hemoproteins engaged in the oxidative biotransformation of various endo- and xenobiotics. Of these, human CYP3A4 is the most dominant liver enzyme, accounting for >30% of the hepatic microsomal P450 complement, and responsible for the oxidative metabolism of over 50% of clinically relevant drugs (1). In common with all the other ER-bound P450s, CYP3A4 is a monotopic protein with its N-terminal ≈33-residue domain embedded in the ER membrane with the bulk of its structure in the cytosol. Our in vivo studies of the heterologously expressed CYP3A4 in the yeast Saccharomyces cerevisiae as well as of its rat liver CYP3A2/3A23 orthologs in primary hepatocytes have revealed that human and rat liver CYPs 3A are turned over via ubiquitin (Ub)-dependent proteasomal degradation (UPD) (2–8). Thus, CYPs 3A represent excellent prototypic substrates of ER-associated degradation (ERAD), specifically of the ERAD-C pathway (6–11). Consistent with this CYP3A ERAD process, our studies of in vivo and/or in vitro reconstituted systems have led us to conclude that CYPs 3A are ubiquitinated by the UBC7/gp78 Ub-ligase complex and recruited by the p97-Npl4-Ufd1 complex before their degradation by the 26 S proteasome (4–8, 12). Because all these processes are energy-dependent, it is not surprising that in vitro reconstitution of CYP3A4 UPD requires ATP. However, inclusion of γ-S-[³²P]ATP in an in vitro reconstituted CYP3A4 ubiquitination system catalyzed by rat liver cytosolic fraction II (FII) resulted in CYP3A4 protein phosphorylation, i.e. γ-[³²P]phosphoryl transfer onto CYP3A4 target residues (13, 14). This phosphorylation was enhanced after cumene hydroperoxide (CuOOH)-mediated CYP3A4 inactivation. The physiological role, if any, of this CYP3A4 post-translational modification is unclear.CYP3A4 is not the only P450 that is phosphorylated. Since the in vitro phosphorylation of a hepatic P450 (CYP2B4) by cAMP-dependent protein kinase A (PKA) was first described (15), various P450s, particularly those belonging to the subfamily 2, were documented to be phosphorylated in cell-free systems, hepatocyte incubations, and intact animals (16–32). Common features of such P450 phosphorylation were the presence of a cytosolically exposed PKA recognition sequence (RRXS) with the Ser residue as the exclusive kinase target, and the ensuing loss of prosthetic heme, conversion to the inactive P420 species, and consequent dramatic functional inactivation (15–20). Studies in intact rats also identified CYPs 3A and 2C6 as kinase targets (21). Although both these P450s lack the hallmark PKA recognition sequence, apparently they possess secondary PKA targeting sequences or are phosphorylated by other protein kinases such as PKC. Indeed, in vitro studies revealed that P450s were phosphorylated in an isoform-dependent manner by either PKA or PKC, except for CYP2B1, which was heavily phosphorylated by both (20). Over the years since this particular post-translational P450 modification was recognized, it has been assigned various functional roles (17, 29–33). Among these, as first proposed by Taniguchi et al. (16) and later explored both by Eliasson et al. (23–26) and us (13, 14), P450 phosphorylation served as a marker for its degradation. Accordingly, the phosphorylation of CYP2E1Ser¹²⁹ and CYP3A1Ser³⁹³ by a microsomal cAMP-dependent protein kinase has been proposed to predispose these P450s but not the similarly phosphorylated CYP2B1 to proteolytic degradation by an integral ER Mg²⁺-ATP-activated serine protease (23–27). However, heterologous expression of CYP2E1S129A/S129G site-directed mutants in COS7 cells apparently had no effect on its relative stability thereby revealing that if CYP2E1 phosphorylation is important for its degradation (34, 35), then alternate Ser/Thr residues (i.e. in plausible secondary PKA recognition sites, Lys-Lys-Ser²⁰⁹-Lys and Lys-Lys-Ser⁴⁴⁹-Ala) may be recruited.On the other hand, on the basis of rapid phosphorylation of CuOOH-inactivated CYP3A4 that precedes its ubiquitination and 26 S proteasomal degradation in an in vitro liver cytosolic FII-catalyzed system, we have proposed that CYP3A4 phosphorylation was essential for targeting it to proteins participating in its UPD/ERAD (13). Indeed, several examples of similar phosphorylation for targeting proteins to UPD exist, of which IκBα phosphorylation is the most notable and perhaps the best documented (36–47; see “Discussion”).Our in vitro studies with specific kinase inhibitors as probes identified both PKC and PKA as the major FII kinases responsible for CYP3A4 phosphorylation (14). Indeed, in vitro model studies of CYP3A4 with PKC as the kinase, coupled with lysylendopeptidase C (Lys-C) digestion of the phosphorylated protein and liquid chromatography-tandem mass spectrometric (LC-MS/MS) analyses of the Lys-C digests, identified two PKC-phosphorylated CYP3A4 peptides ²⁵⁸ESRLEDpTQK²⁶⁶ and ⁴¹⁴FLPERFpSK⁴²¹ unambiguously phosphorylated at Thr²⁶⁴ and Ser⁴²⁰ (14). These same residues were also phosphorylated in corresponding studies with PKA.⁴ Furthermore, although both native and CuOOH-inactivated CYP3A4 were phosphorylated at Thr²⁶⁴, Ser⁴²⁰ phosphorylation was particularly enhanced after CuOOH-mediated CYP3A4 inactivation (14). Corresponding studies of CuOOH-inactivated CYP3A4 using rat liver cytosolic FII as the source of the kinase(s), revealed ³²P phosphorylation of both these peptides as well as that of an additional CYP3A4 peptide ⁴⁷⁷LS(p)LGGLLQPEKPVVLK⁴⁹². Unlike the unambiguous mass spectrometric identification of Thr²⁶⁴ and Ser⁴²⁰ as the phosphorylated CYP3A4 residues, the phosphorylation of Ser⁴⁷⁸, the only plausible phosphorylatable residue in this ³²P-labeled peptide, was not similarly established. Nevertheless, the predominant phosphorylation of Thr²⁶⁴ in native CYP3A4 (14), but of two additional residues in the CuOOH-inactivated enzyme, is consistent with the inactivation-induced structural unraveling of this enzyme with exposure of otherwise concealed and/or kinase-inaccessible domains (48). Such unraveling of CYP3A4 protein stems from the irreversible modification of its active site by fragments generated from CuOOH-mediated oxidative destruction of its prosthetic heme (49). In this study, using mass spectrometric analyses of Lys-C digests of FII-phosphorylated CYP3A4, we have provided unambiguous evidence that in addition to Thr²⁶⁴ and Ser⁴²⁰, Ser⁴⁷⁸ is indeed phosphorylated. More importantly, through alanine-scanning mutagenesis of these three residues, we now document that although neither the structural conformation nor the catalytic function of this triple CYP3A4T264A/S420A/S478A mutant is altered, its degradation after heterologous expression in S. cerevisiae is significantly impaired. This is also true of CYP3A4T264A/S420A/S478A mutant degradation in human embryonic kidney (HEK293T) cells. Furthermore, using an in vitro reconstituted CYP3A4 ubiquitination system, catalyzed by human Ub-conjugating E2 enzyme UBC7 and integral ER protein gp78 as the E3 Ub ligase (12), we document that PKA/PKC-mediated phosphorylation of the wild type CYP3A4 (CYP3A4wt) considerably enhanced its UBC7/gp78-mediated ubiquitination. Together these findings reveal the critical importance of CYP3A4 phosphorylation at these residues for its UPD and suggest a direct link between phosphorylation and its ubiquitination and degradation.     

Density-enhanced Phosphatase 1 Regulates Phosphorylation of Tight Junction Proteins and Enhances Barrier Function of Epithelial Cells

Cell-cell adhesion is a dynamic process that can activate multiple signaling pathways. These signaling pathways can be regulated through reversible tyrosine phosphorylation events. The level of tyrosine phosphorylation of junctional proteins reflects the balance between protein-tyrosine kinase and protein-tyrosine phosphatase activity. The receptor-tyrosine phosphatase DEP-1 (CD148/PTP-η) has been implicated in cell growth and differentiation as well as in regulating phosphorylation of junctional proteins. However, the role of DEP-1 in regulating tight junction phosphorylation and the integrity of cell-cell junctions is still under investigation. In this study, we used a catalytically dead substrate-trapping mutant of DEP-1 to identify potential substrates at cell-cell junctions. We have shown that in epithelial cells the trapping mutant of DEP-1 interacts with the tight junction proteins occludin and ZO-1 in a tyrosine phosphorylation-dependent manner. In contrast, PTP-PEST, Shp2, and PTPμ did not interact with these proteins, suggesting that the interaction of DEP-1 with occludin and ZO-1 is specific. In addition, occludin and ZO-1 were dephosphorylated by DEP-1 but not these other phosphatases in vitro. Overexpression of DEP-1 increased barrier function as measured by transepithelial electrical resistance and also reduced paracellular flux of fluorescein isothiocyanate-dextran following a calcium switch. Reduced DEP-1 expression by small interfering RNA had a small but significant increase in junction permeability. These data suggest that DEP-1 can modify the phosphorylation state of tight junction proteins and play a role in regulating permeability.Tight junctions are the most apical of junctions formed by epithelia and provide a regulated barrier to paracellular transport of ions, solutes, macromolecules, and even other cells. In addition, tight junctions act as a “fence” within the plane of the membrane, dividing the apical and basolateral domains of polarized epithelial cells. These junctions play an important role in the regulation of multiple cellular processes including cell differentiation, proliferation, and polarity (for reviews see Refs. 1 and 2). Functional tight junctions are characterized by the presence of membrane spanning proteins (claudins, occludin, and JAMs), which interact with cytoplasmic proteins (AF-6 and ZO-1, -2, -3), regulating assembly and maintenance of tight junctions. Occludin spans the membrane four times and was the first transmembrane component of the tight junction to be identified (3). It has two extracellular regions, an intracellular loop, as well as both an N- and C-terminal cytoplasmic tail (3). The C-terminal tail of occludin binds directly to the ZO family of proteins, which link the protein complex to the actin cytoskeleton (4–8). The long C-terminal domain is rich in serine, threonine, and tyrosine residues (9). In fact, several kinases and phosphatases interact with and modulate the phosphorylation state of tight junction proteins (10–14). Serine and threonine phosphorylation of occludin is abundant in epithelia with intact junctions, whereas tyrosine phosphorylation is undetectable (15). However, tyrosine phosphorylation of occludin is associated with a decrease in transepithelial electrical resistance (TER)² (16, 17) and loss of protein localization at the tight junction (18). Increases in tyrosine phosphorylation of occludin and ZO-1 result in the dissociation of the occludin-ZO-1 complex and reduces the localization at the tight junction of these proteins (12, 19). These data suggest that the phosphorylation state of tight junction proteins can affect the integrity of the tight junction complex and therefore the integrity of the tight junction itself. Both serine and threonine kinases and phosphatases bind to and act on TJ proteins (reviewed in Ref. 20). Previous studies have identified that c-Src and c-Yes are protein-tyrosine kinases, which act on the TJ, however, to date no protein-tyrosine phosphatases have been specifically characterized as acting on TJ proteins (11–13, 21).Similarly, the other major junction of epithelia, the adherens junction (AJ), is also regulated by tyrosine phosphorylation. Increased tyrosine phosphorylation of the AJ decreases the stability of the cadherin-catenin complex, disrupting the association with the cytoskeleton and reducing junctional integrity (22–24). Therefore, these studies suggest that maintenance of junctional integrity for both the TJ and AJ is regulated in part by reversible tyrosine phosphorylation that results from a competing balance of protein-tyrosine kinase and protein-tyrosine phosphatase (PTP) activity. Several PTPs have been localized to AJs and shown to bind components of the cadherin-catenin complex. The PTPs in AJs include receptor-PTPs (PTPμ, DEP-1, and vascular endothelial-PTP), as well as cytosolic PTPs (PTP1B and Shp-2) (25–30). The high concentration of PTPs at cell-cell junctions indicates the importance of maintaining low levels of tyrosine phosphorylation except when the junctions need to be remodeled or disassembled.DEP-1 (density-enhanced phosphatase-1) is a receptor PTP that was first cloned from a human cDNA library and named based on the observation that its expression was elevated with increasing cell density (31). Also known as PTP-η, PTPRJ, and CD148, DEP-1 is comprised of an extracellular domain of eight fibronectin type III repeats, a transmembrane domain, and a single cytoplasmic catalytic domain. The protein is ubiquitously expressed (32), indicating its potential involvement in a large number of diverse signaling pathways. DEP-1 is involved in regulating the differentiation of epithelial cells (33–36), as well as controlling cell growth and adhesion (33, 34). In addition, DEP-1 is able to attenuate the cellular response to growth factors through the preferential dephosphorylation of several growth factor receptors, suggesting that DEP-1 can selectively dephosphorylate certain tyrosines to more finely control signaling (37–41).In addition to its role in proliferation and differentiation, DEP-1 localizes to areas of cell-cell adhesion in endothelial and epithelial cells, overlapping with the AJ marker vascular endothelial-cadherin in endothelia (42). Interaction with p120 catenin as well as other members of the catenin family also supports the hypothesis that DEP-1 plays a role in regulating AJ protein phosphorylation (27, 40). In the current study, we investigated whether adjacent tight junction proteins are also substrates of DEP-1. We now demonstrate that the substrate-trapping mutant of DEP-1 interacts with the tight junction proteins occludin and ZO-1. The association of DEP-1 with occludin and ZO-1 is specific to DEP-1 and not other phosphatases tested. In addition, DEP-1 is able to dephosphorylate occludin and ZO-1 indicating that these tight junction proteins are substrates of DEP-1. Furthermore, increased expression of DEP-1 enhances barrier function as junctions reform following a calcium switch and loss of DEP-1 levels increased the permeability of a stable epithelial monolayer. Together these results indicate that ZO-1 and occludin are substrates of DEP-1 and imply a role for DEP-1 in influencing the phosphorylation state of tight junction proteins and junction permeability.     

Phosphorylation of p53 Is Regulated by TPX2-Aurora A in Xenopus Oocytes

p53 is an important tumor suppressor regulating the cell cycle at multiple stages in higher vertebrates. The p53 gene is frequently deleted or mutated in human cancers, resulting in loss of p53 activity. This leads to centrosome amplification, aneuploidy, and tumorigenesis, three phenotypes also observed after overexpression of the oncogenic kinase Aurora A. Accordingly, recent studies have focused on the relationship between these two proteins. p53 and Aurora A have been reported to interact in mammalian cells, but the function of this interaction remains unclear. We recently reported that Xenopus p53 can inhibit Aurora A activity in vitro but only in the absence of TPX2. Here we investigate the interplay between Xenopus Aurora A, TPX2, and p53 and show that newly synthesized TPX2 is required for nearly all Aurora A activation and for full p53 synthesis and phosphorylation in vivo during oocyte maturation. In vitro, phosphorylation mediated by Aurora A targets serines 129 and 190 within the DNA binding domain of p53. Glutathione S-transferase pull-down studies indicate that the interaction occurs via the p53 transactivation domain and the Aurora A catalytic domain around the T-loop. Our studies suggest that targeting of TPX2 might be an effective strategy for specifically inhibiting the phosphorylation of Aurora A substrates, including p53.Aurora A is an oncogenic protein kinase that is active in mitosis and plays important roles in spindle assembly and centrosome function (1). Overexpression of either human or Xenopus Aurora A transforms mammalian cells, but only when the p53 pathway is altered (2–4). Aurora A is localized on centrosomes during mitosis, and overexpression of the protein leads to centrosome amplification and aneuploidy (2, 3, 5, 6), two likely contributors to genomic instability (7, 8). Because of its oncogenic potential and amplification in human tumors, considerable attention has been focused on the mechanism of Aurora A activation in mitosis. Evidence from several laboratories indicates that activation occurs as a result of phosphorylation of a threonine residue in the T-loop of the kinase (4, 9, 10). Purification of Aurora A-activating activity from M phase Xenopus egg extracts led to an apparent activation mechanism in which autophosphorylation at the T-loop is stimulated by binding of the targeting protein for Xklp2 (TPX2) (11–14). On the other hand, it has been shown that Aurora A activity can be inhibited by interaction with several proteins, including PP1 (protein phosphatase 1), AIP (Aurora A kinase-interacting protein), and, more recently, p53 (9, 15–17).p53 is a well known tumor suppressor able to drive cell cycle arrest, apoptosis, or senescence when DNA is damaged or cell integrity is threatened (18, 19). In human cancers, the p53 gene is frequently deleted or mutated, leading to inactivation of p53 functions (20). p53 protein is almost undetectable in “normal cells,” mainly due to its instability. Indeed, during a normal cell cycle, p53 associates with Mdm2 in the nucleus and thereafter undergoes nuclear exclusion, allowing its ubiquitination and subsequent degradation (21). In cells under stress, p53 is stabilized through the disruption of its interaction with Mdm2 (21), leading to p53 accumulation in the nucleus and triggering different responses, as described above.Although p53 has mostly been characterized as a nuclear protein, it has also been shown to localize on centrosomes (22–24) and regulate centrosome duplication (23, 24). Centrosomes are believed to act as scaffolds that concentrate many regulatory molecules involved in signal transduction, including multiple protein kinases (25). Thus, centrosomal localization of p53 might be important for its own regulation by phosphorylation/dephosphorylation, and one of its regulators could be the mitotic kinase Aurora A. Indeed, phenotypes associated with the misexpression of these two proteins are very similar. For example, overexpression of Aurora A kinase leads to centrosome amplification, aneuploidy, and tumorigenesis, and the same effects are often observed after down-regulation of p53 transactivation activity or deletion/mutation of its gene (26, 27).Several recent studies performed in mammalian models show interplay between p53 and Aurora A, with each protein having the ability to inhibit the other, depending on the stage of the cell cycle and the stress level of the cell (17, 28, 29). These studies reported that p53 is a substrate of Aurora A, and serines 215 and 315 were demonstrated to be the two major Aurora A phosphorylation sites in human p53 in vitro and in vivo. Phosphorylation of Ser-215 within the DNA binding domain of human p53 inhibited both p53 DNA binding and transactivation activities (29). Recently, our group showed that Xenopus p53 is able to inhibit Aurora A kinase activity in vitro, but this inhibitory effect can be suppressed by prior binding of Aurora A to TPX2 (9). Contrary to somatic cells, where p53 is nuclear, unstable, and expressed at a very low level, p53 is highly expressed in the cytoplasm of Xenopus oocytes and stable until later stages of development (30, 31). The high concentration of both p53 and Aurora A in the oocyte provided a suitable basis for investigating p53-Aurora A interaction and also evaluating Xenopus p53 as a substrate of Aurora A.     

Effects of Combined Phosphorylation at Ser-617 and Ser-1179 in Endothelial Nitric-oxide Synthase on EC50(Ca2+) Values for Calmodulin Binding and Enzyme Activation

We have investigated the possible biochemical basis for enhancements in NO production in endothelial cells that have been correlated with agonist- or shear stress-evoked phosphorylation at Ser-1179. We have found that a phosphomimetic substitution at Ser-1179 doubles maximal synthase activity, partially disinhibits cytochrome c reductase activity, and lowers the EC₅₀(Ca²⁺) values for calmodulin binding and enzyme activation from the control values of 182 ± 2 and 422 ± 22 nm to 116 ± 2 and 300 ± 10 nm. These are similar to the effects of a phosphomimetic substitution at Ser-617 (Tran, Q. K., Leonard, J., Black, D. J., and Persechini, A. (2008) Biochemistry 47, 7557–7566). Although combining substitutions at Ser-617 and Ser-1179 has no additional effect on maximal synthase activity, cooperativity between the two substitutions completely disinhibits reductase activity and further reduces the EC₅₀(Ca²⁺) values for calmodulin binding and enzyme activation to 77 ± 2 and 130 ± 5 nm. We have confirmed that specific Akt-catalyzed phosphorylation of Ser-617 and Ser-1179 and phosphomimetic substitutions at these positions have similar functional effects. Changes in the biochemical properties of eNOS produced by combined phosphorylation at Ser-617 and Ser-1179 are predicted to substantially increase synthase activity in cells at a typical basal free Ca²⁺ concentration of 50–100 nm.The nitric-oxide synthases catalyze formation of NO and l-citrulline from l-arginine and O₂, with NADPH as the electron donor (1). The role of NO generated by endothelial nitricoxide synthase (eNOS)² in the regulation of smooth muscle tone is well established and was the first of several physiological roles for this small molecule that have so far been identified (2). The nitric-oxide synthases are homodimers of 130–160-kDa subunits. Each subunit contains a reductase and oxygenase domain (1). A significant difference between the reductase domains in eNOS and nNOS and the homologous P450 reductases is the presence of inserts in these synthase isoforms that appear to maintain them in their inactive states (3, 4). A calmodulin (CaM)-binding domain is located in the linker that connects the reductase and oxygenase domains, and the endothelial and neuronal synthases both require Ca²⁺ and exogenous CaM for activity (5, 6). When CaM is bound, it somehow counteracts the effects of the autoinhibitory insert(s) in the reductase. The high resolution structure for the complex between (Ca²⁺)₄-CaM and the isolated CaM-binding domain from eNOS indicates that the C-ter and N-ter lobes of CaM, which each contain a pair of Ca²⁺-binding sites, enfold the domain, as has been observed in several other such CaM-peptide complexes (7). Consistent with this structure, investigations of CaM-dependent activation of the neuronal synthase suggest that both CaM lobes must participate (8, 9).Bovine eNOS can be phosphorylated in endothelial cells at Ser-116, Thr-497, Ser-617, Ser-635, and Ser-1179 (10–12). There are equivalent phosphorylation sites in the human enzyme (10–12). Phosphorylation of the bovine enzyme at Thr-497, which is located in the CaM-binding domain, blocks CaM binding and enzyme activation (7, 11, 13, 14). Ser-116 can be basally phosphorylated in cells (10, 11, 13, 15), and dephosphorylation of this site has been correlated with increased NO production (13, 15). However, it has also been reported that a phosphomimetic substitution at this position has no effect on enzyme activity measured in vitro (13). Ser-1179 is phosphorylated in response to a variety of stimuli, and this has been reliably correlated with enhanced NO production in cells (10, 11). Indeed, NO production is elevated in transgenic endothelium expressing an eNOS mutant containing an S1179D substitution, but not in tissue expressing an S1179A mutant (16). Shear stress or insulin treatment is correlated with Akt-catalyzed phosphorylation of Ser-1179 in endothelial cells, and this is correlated with increased NO production in the absence of extracellular Ca²⁺ (17–19). Akt-catalyzed phosphorylation or an S1179D substitution has also been correlated with increased synthase activity in cell extracts at low intracellular free [Ca²⁺] (17). Increased NO production has also been observed in cells expressing an eNOS mutant containing an S617D substitution, and physiological stimuli such as shear-stress, bradykinin, VEGF, and ATP appear to stimulate Akt-catalyzed phosphorylation of Ser-617 and Ser-1179 (12, 13, 20). Although S617D eNOS has been reported to have the same maximum activity in vitro as the wild type enzyme (20), in our hands an S617D substitution increases the maximal CaM-dependent synthase activity of purified mutant enzyme ∼2-fold, partially disinhibits reductase activity, and reduces the EC₅₀(Ca²⁺) values for CaM binding and enzyme activation (21).In this report, we describe the effects of a phosphomimetic Asp substitution at Ser-1179 in eNOS on the Ca²⁺ dependence of CaM binding and CaM-dependent activation of reductase and synthase activities. We also describe the effects on these properties of combining this substitution with one at Ser-617. Finally, we demonstrate that Akt-catalyzed phosphorylation and Asp substitutions at Ser-617 and Ser-1179 have similar functional effects. Our results suggest that phosphorylation of eNOS at Ser-617 and Ser-1179 can substantially increase synthase activity in cells at a typical basal free Ca²⁺ concentration of 50–100 nm, while single phosphorylations at these sites produce smaller activity increases, and can do so only at higher free Ca²⁺ concentrations.     

Bortezomib Overcomes Tumor Necrosis Factor-related Apoptosis-inducing Ligand Resistance in Hepatocellular Carcinoma Cells in Part through the Inhibition of the Phosphatidylinositol 3-Kinase/Akt Pathway

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However, many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis. Comparing the molecular change in HCC cells treated with these agents, we found that down-regulation of phospho-Akt (P-Akt) played a key role in mediating TRAIL sensitization of bortezomib. The first evidence was that bortezomib down-regulated P-Akt in a dose- and time-dependent manner in TRAIL-treated HCC cells. Second, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor, also sensitized resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells. Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in bortezomib-treated cells, and PP2A knockdown by small interference RNA also reduced apoptosis induced by the combination of TRAIL and bortezomib, indicating that PP2A may be important in mediating the effect of bortezomib on TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at clinically achievable concentrations in hepatocellular carcinoma cells, and this effect is mediated at least partly via inhibition of the PI3K/Akt pathway.Hepatocellular carcinoma (HCC)² is currently the fifth most common solid tumor worldwide and the fourth leading cause of cancer-related death. To date, surgery is still the only curative treatment but is only feasible in a small portion of patients (1). Drug treatment is the major therapy for patients with advanced stage disease. Unfortunately, the response rate to traditional chemotherapy for HCC patients is unsatisfactory (1). Novel pharmacological therapy is urgently needed for patients with advanced HCC. In this regard, the approval of sorafenib might open a new era of molecularly targeted therapy in the treatment of HCC patients.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a type II transmembrane protein and a member of the TNF family, is a promising anti-tumor agent under clinical investigation (2). TRAIL functions by engaging its receptors expressed on the surface of target cells. Five receptors specific for TRAIL have been identified, including DR4/TRAIL-R1, DR5/TRAIL-R2, DcR1, DcR2, and osteoprotegerin. Among TRAIL receptors, only DR4 and DR5 contain an effective death domain that is essential to formation of death-inducing signaling complex (DISC), a critical step for TRAIL-induced apoptosis. Notably, the trimerization of the death domains recruits an adaptor molecule, Fas-associated protein with death domain (FADD), which subsequently recruits and activates caspase-8. In type I cells, activation of caspase-8 is sufficient to activate caspase-3 to induce apoptosis; however, in another type of cells (type II), the intrinsic mitochondrial pathway is essential for apoptosis characterized by cleavage of Bid and release of cytochrome c from mitochondria, which subsequently activates caspase-9 and caspase-3 (3).Although TRAIL induces apoptosis in malignant cells but sparing normal cells, some tumor cells are resistant to TRAIL-induced apoptosis. Mechanisms responsible for the resistance include receptors and intracellular resistance. Although the cell surface expression of DR4 or DR5 is absolutely required for TRAIL-induced apoptosis, tumor cells expressing these death receptors are not always sensitive to TRAIL due to intracellular mechanisms. For example, the cellular FLICE-inhibitory protein (c-FLIP), a homologue to caspase-8 but without protease activity, has been linked to TRAIL resistance in several studies (4, 5). In addition, inactivation of Bax, a proapoptotic Bcl-2 family protein, resulted in resistance to TRAIL in MMR-deficient tumors (6, 7), and reintroduction of Bax into Bax-deficient cells restored TRAIL sensitivity (8), indicating that the Bcl-2 family plays a critical role in intracellular mechanisms for resistance of TRAIL.Bortezomib, a proteasome inhibitor approved clinically for multiple myeloma and mantle cell lymphoma, has been investigated intensively for many types of cancer (9). Accumulating studies indicate that the combination of bortezomib and TRAIL overcomes the resistance to TRAIL in various types of cancer, including acute myeloid leukemia (4), lymphoma (10–13), prostate (14–17), colon (15, 18, 19), bladder (14, 16), renal cell carcinoma (20), thyroid (21), ovary (22), non-small cell lung (23, 24), sarcoma (25), and HCC (26, 27). Molecular targets responsible for the sensitizing effect of bortezomib on TRAIL-induced cell death include DR4 (14, 27), DR5 (14, 20, 22–23, 28), c-FLIP (4, 11, 21–23, 29), NF-κB (12, 24, 30), p21 (16, 21, 25), and p27 (25). In addition, Bcl-2 family also plays a role in the combinational effect of bortezomib and TRAIL, including Bcl-2 (10, 21), Bax (13, 22), Bak (27), Bcl-xL (21), Bik (18), and Bim (15).Recently, we have reported that Akt signaling is a major molecular determinant in bortezomib-induced apoptosis in HCC cells (31). In this study, we demonstrated that bortezomib overcame TRAIL resistance in HCC cells through inhibition of the PI3K/Akt pathway.     

Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5

NHE5 is a brain-enriched Na⁺/H⁺ exchanger that dynamically shuttles between the plasma membrane and recycling endosomes, serving as a mechanism that acutely controls the local pH environment. In the current study we show that secretory carrier membrane proteins (SCAMPs), a group of tetraspanning integral membrane proteins that reside in multiple secretory and endocytic organelles, bind to NHE5 and co-localize predominantly in the recycling endosomes. In vitro protein-protein interaction assays revealed that NHE5 directly binds to the N- and C-terminal cytosolic extensions of SCAMP2. Heterologous expression of SCAMP2 but not SCAMP5 increased cell-surface abundance as well as transporter activity of NHE5 across the plasma membrane. Expression of a deletion mutant lacking the SCAMP2-specific N-terminal cytosolic domain, and a mini-gene encoding the N-terminal extension, reduced the transporter activity. Although both Arf6 and Rab11 positively regulate NHE5 cell-surface targeting and NHE5 activity across the plasma membrane, SCAMP2-mediated surface targeting of NHE5 was reversed by dominant-negative Arf6 but not by dominant-negative Rab11. Together, these results suggest that SCAMP2 regulates NHE5 transit through recycling endosomes and promotes its surface targeting in an Arf6-dependent manner.Neurons and glial cells in the central and peripheral nervous systems are especially sensitive to perturbations of pH (1). Many voltage- and ligand-gated ion channels that control membrane excitability are sensitive to changes in cellular pH (1-3). Neurotransmitter release and uptake are also influenced by cellular and organellar pH (4, 5). Moreover, the intra- and extracellular pH of both neurons and glia are modulated in a highly transient and localized manner by neuronal activity (6, 7). Thus, neurons and glia require sophisticated mechanisms to finely tune ion and pH homeostasis to maintain their normal functions.Na⁺/H⁺ exchangers (NHEs)³ were originally identified as a class of plasma membrane-bound ion transporters that exchange extracellular Na⁺ for intracellular H⁺, and thereby regulate cellular pH and volume. Since the discovery of NHE1 as the first mammalian NHE (8), eight additional isoforms (NHE2-9) that share 25-70% amino acid identity have been isolated in mammals (9, 10). NHE1-5 commonly exhibit transporter activity across the plasma membrane, whereas NHE6-9 are mostly found in organelle membranes and are believed to regulate organellar pH in most cell types at steady state (11). More recently, NHE10 was identified in human and mouse osteoclasts (12, 13). However, the cDNA encoding NHE10 shares only a low degree of sequence similarity with other known members of the NHE gene family, raising the possibility that this sodium-proton exchanger may belong to a separate gene family distantly related to NHE1-9 (see Ref. 9).NHE gene family members contain 12 putative transmembrane domains at the N terminus followed by a C-terminal cytosolic extension that plays a role in regulation of the transporter activity by protein-protein interactions and phosphorylation. NHEs have been shown to regulate the pH environment of synaptic nerve terminals and to regulate the release of neurotransmitters from multiple neuronal populations (14-16). The importance of NHEs in brain function is further exemplified by the findings that spontaneous or directed mutations of the ubiquitously expressed NHE1 gene lead to the progression of epileptic seizures, ataxia, and increased mortality in mice (17, 18). The progression of the disease phenotype is associated with loss of specific neuron populations and increased neuronal excitability. However, NHE1-null mice appear to develop normally until 2 weeks after birth when symptoms begin to appear. Therefore, other mechanisms may compensate for the loss of NHE1 during early development and play a protective role in the surviving neurons after the onset of the disease phenotype.NHE5 was identified as a unique member of the NHE gene family whose mRNA is expressed almost exclusively in the brain (19, 20), although more recent studies have suggested that NHE5 might be functional in other cell types such as sperm (21, 22) and osteosarcoma cells (23). Curiously, mutations found in several forms of congenital neurological disorders such as spinocerebellar ataxia type 4 (24-26) and autosomal dominant cerebellar ataxia (27-29) have been mapped to chromosome 16q22.1, a region containing NHE5. However, much remains unknown as to the molecular regulation of NHE5 and its role in brain function.Very few if any proteins work in isolation. Therefore identification and characterization of binding proteins often reveal novel functions and regulation mechanisms of the protein of interest. To begin to elucidate the biological role of NHE5, we have started to explore NHE5-binding proteins. Previously, β-arrestins, multifunctional scaffold proteins that play a key role in desensitization of G-protein-coupled receptors, were shown to directly bind to NHE5 and promote its endocytosis (30). This study demonstrated that NHE5 trafficking between endosomes and the plasma membrane is regulated by protein-protein interactions with scaffold proteins. More recently, we demonstrated that receptor for activated C-kinase 1 (RACK1), a scaffold protein that links signaling molecules such as activated protein kinase C, integrins, and Src kinase (31), directly interacts with and activates NHE5 via integrin-dependent and independent pathways (32). These results further indicate that NHE5 is partly associated with focal adhesions and that its targeting to the specialized microdomain of the plasma membrane may be regulated by various signaling pathways.Secretory carrier membrane proteins (SCAMPs) are a family of evolutionarily conserved tetra-spanning integral membrane proteins. SCAMPs are found in multiple organelles such as the Golgi apparatus, trans-Golgi network, recycling endosomes, synaptic vesicles, and the plasma membrane (33, 34) and have been shown to play a role in exocytosis (35-38) and endocytosis (39). Currently, five isoforms of SCAMP have been identified in mammals. The extended N terminus of SCAMP1-3 contain multiple Asn-Pro-Phe (NPF) repeats, which may allow these isoforms to participate in clathrin coat assembly and vesicle budding by binding to Eps15 homology (EH)-domain proteins (40, 41). Further, SCAMP2 was shown recently to bind to the small GTPase Arf6 (38), which is believed to participate in traffic between the recycling endosomes and the cell surface (42, 43). More recent studies have suggested that SCAMPs bind to organellar membrane type NHE7 (44) and the serotonin transporter SERT (45) and facilitate targeting of these integral membrane proteins to specific intracellular compartments. We show in the current study that SCAMP2 binds to NHE5, facilitates the cell-surface targeting of NHE5, and elevates Na⁺/H⁺ exchange activity at the plasma membrane, whereas expression of a SCAMP2 deletion mutant lacking the N-terminal domain containing the NPF repeats suppresses the effect. Further we show that this activity of SCAMP2 requires an active form of a small GTPase Arf6, but not Rab11. We propose a model in which SCAMPs bind to NHE5 in the endosomal compartment and control its cell-surface abundance via an Arf6-dependent pathway.     

Strain-induced Proliferation Requires the Phosphatidylinositol 3-Kinase/AKT/Glycogen Synthase Kinase Pathway

The intestinal epithelium is repetitively deformed by shear, peristalsis, and villous motility. Such repetitive deformation stimulates the proliferation of intestinal epithelial cells on collagen or laminin substrates via ERK, but the upstream mediators of this effect are poorly understood. We hypothesized that the phosphatidylinositol 3-kinase (PI3K)/AKT cascade mediates this mitogenic effect. PI3K, AKT, and glycogen synthase kinase-3β (GSK-3β) were phosphorylated by 10 cycles/min strain at an average 10% deformation, and pharmacologic blockade of these molecules or reduction by small interfering RNA (siRNA) prevented the mitogenic effect of strain in Caco-2 or IEC-6 intestinal epithelial cells. Strain MAPK activation required PI3K but not AKT. AKT isoform-specific siRNA transfection demonstrated that AKT2 but not AKT1 is required for GSK-3β phosphorylation and the strain mitogenic effect. Furthermore, overexpression of AKT1 or an AKT chimera including the PH domain and hinge region of AKT2 and the catalytic domain and C-tail of AKT1 prevented strain activation of GSK-3β, but overexpression of AKT2 or a chimera including the PH domain and hinge region of AKT1 and the catalytic domain and C-tail of AKT2 did not. These data delineate a role for PI3K, AKT2, and GSK-3β in the mitogenic effect of strain. PI3K is required for both ERK and AKT2 activation, whereas AKT2 is sequentially required for GSK-3β. Furthermore, AKT2 specificity requires its catalytic domain and tail region. Manipulating this pathway may prevent mucosal atrophy and maintain the mucosal barrier in conditions such as ileus, sepsis, and prolonged fasting when peristalsis and villous motility are decreased and the mucosal barrier fails.Mechanical forces are part of the normal intestinal epithelial environment. Numerous different forces deform these cells including shear stress from endoluminal chyme, bowel peristalsis, and villous motility (1, 2). During normal bowel function the mucosa is subjected to injury that must be repaired to maintain the mucosal barrier (3, 4). Deformation patterns of the bowel are altered in conditions such as prolonged fasting, post-surgical ileus, and sepsis states, resulting in profoundly reduced mucosal deformation. When such states are prolonged, proliferation slows, the mucosa becomes atrophic, and bacterial translocation may ensue as the mucosal barrier of the gut breaks down (5–7).In vitro, repetitive deformation is trophic for intestinal epithelial cells (8) cultured on type I or type IV collagen or laminin. Human Caco-2 intestinal epithelial cells (9), non-transformed rat IEC-6 intestinal epithelial cells (10), and primary human intestinal epithelial cells isolated from surgical specimens (11) proliferate more rapidly in response to cyclic strain (12) unless substantial quantities of fibronectin are added to the media or matrix (11) to mimic the acute phase reaction of acute or chronic inflammation and injury. Cyclic strain also stimulates proliferation in HCT 116 colon cancer cells (13) and differentiation of Caco-2 cells cultured on a collagen substrate (9). This phenomenon has also been observed in vivo (14). Thus, repetitive deformation may help to maintain the normal homeostasis of the gut mucosa under non-inflammatory conditions. Previous work in our laboratory has implicated Src, focal adhesion kinase, and the mitogen-activated protein kinase (MAPK)² extracellular signal-related kinase (ERK) in the mitogenic effect of strain (10). Although p38 is also activated in Caco-2 cells subjected to cyclic strain on a collagen matrix, its activity is not required for the mitogenic effect of strain (12).Although often the PI3K/AKT pathway is thought of as a parallel pathway to the MAPK, this is not always the case. Protein kinase C isoenzymes differentially modulate thrombin effect on MAPK-dependent retinal pigment epithelial cell (RPE) proliferation, and it has been shown that PI3K or AKT inhibition prevented thrombin-induced ERK activation and RPE proliferation (15).PI3K, AKT, and glycogen synthase kinase (GSK), a downstream target of AKT (16), have been implemented in intestinal epithelial cell proliferation in numerous cell systems not involving strain (17–19) including uncontrolled proliferation in gastrointestinal cancers (20–22). Mechanical forces activate this pathway as well. PI3K and AKT are required for increased extracellular pressure to stimulate colon cancer cell adhesion (23), although the pathway by which pressure stimulates colon cancer cells in suspension differs from the response of adherent intestinal epithelial cells to repetitive deformation (24), and GSK is not involved in this effect.³ Repetitive strain also stimulates vascular endothelial cell proliferation via PI3K and AKT (25, 26), whereas respiratory strain stimulates angiogenic responses via PI3K (27). We, therefore, hypothesized that the PI3K/AKT/GSK axis would be involved in the mitogenic effects of repetitive deformation on a collagen matrix.To test this hypothesis, we used the Flexcell apparatus to rhythmically deform Caco-2 intestinal epithelial cells. IEC-6 cells were used to confirm key results. A frequency of 10 cycles per min was used, which is similar in order of magnitude to the frequency that the intestinal mucosa might be deformed by peristalsis or villous motility in vivo (28, 29). Mechanical forces such as repetitive deformation are likely cell-type and frequency-specific, as different cell types respond to different frequencies. Vascular endothelial cells respond to frequencies of 60–80 cycles/min (25), whereas intestinal epithelial cells may actually decrease proliferation in response to frequencies of 5 cycles/min (30). We characterized PI3K, AKT, and GSK phosphorylation with strain, blocked these molecules pharmacologically or by siRNA, and delineated the specificity of the AKT effect using isozyme-specific siRNA and transfection of AKT1/2 chimeras. We also characterized the interaction of this pathway with the activation of ERK by strain, which has previously been implicated in the mitogenic response (12).     

Overexpression of E-cadherin on Melanoma Cells Inhibits Chemokine-promoted Invasion Involving p190RhoGAP/p120ctn-dependent Inactivation of RhoA

Melanoma cells express the chemokine receptor CXCR4 that confers high invasiveness upon binding to its ligand CXCL12. Melanoma cells at initial stages of the disease show reduction or loss of E-cadherin expression, but recovery of its expression is frequently found at advanced phases. We overexpressed E-cadherin in the highly invasive BRO lung metastatic cell melanoma cell line to investigate whether it could influence CXCL12-promoted cell invasion. Overexpression of E-cadherin led to defective invasion of melanoma cells across Matrigel and type I collagen in response to CXCL12. A decrease in individual cell migration directionality toward the chemokine and reduced adhesion accounted for the impaired invasion. A p190RhoGAP-dependent inhibition of RhoA activation was responsible for the impairment in chemokine-stimulated E-cadherin melanoma transfectant invasion. Furthermore, we show that p190RhoGAP and p120ctn associated predominantly on the plasma membrane of cells overexpressing E-cadherin, and that E-cadherin-bound p120ctn contributed to RhoA inactivation by favoring p190RhoGAP-RhoA association. These results suggest that melanoma cells at advanced stages of the disease could have reduced metastatic potency in response to chemotactic stimuli compared with cells lacking E-cadherin, and the results indicate that p190RhoGAP is a central molecule controlling melanoma cell invasion.Cadherins are a family of Ca²⁺-dependent adhesion molecules that mediate cell-cell contacts and are expressed in most solid tissues providing a tight control of morphogenesis (1, 2). Classical cadherins, such as epithelial (E) cadherin, are found in adherens junctions, forming core protein complexes with β-catenin, α-catenin, and p120 catenin (p120ctn). Both β-catenin and p120ctn directly interact with E-cadherin, whereas α-catenin associates with the complex through its binding to β-catenin, providing a link with the actin cytoskeleton (1, 2). E-cadherin is frequently lost or down-regulated in many human tumors, coincident with morphological epithelial to mesenchymal transition and acquisition of invasiveness (3-6).Although melanoma only accounts for 5% of skin cancers, when metastasis starts, it is responsible for 80% of deaths from skin cancers (7). Melanocytes express E-cadherin (8-10), but melanoma cells at early radial growth phase show a large reduction in the expression of this cadherin, and surprisingly, expression has been reported to be partially recovered by vertical growth phase and metastatic melanoma cells (9, 11, 12).Trafficking of cancer cells from primary tumor sites to intravasation into blood circulation and later to extravasation to colonize distant organs requires tightly regulated directional cues and cell migration and invasion that are mediated by chemokines, growth factors, and adhesion molecules (13). Solid tumor cells express chemokine receptors that provide guidance of these cells to organs where their chemokine ligands are expressed, constituting a homing model resembling the one used by immune cells to exert their immune surveillance functions (14). Most solid cancer cells express CXCR4, a receptor for the chemokine CXCL12 (also called SDF-1), which is expressed in lungs, bone marrow, and liver (15). Expression of CXCR4 in human melanoma has been detected in the vertical growth phase and on regional lymph nodes, which correlated with poor prognosis and increased mortality (16, 17). Previous in vivo experiments have provided evidence supporting a crucial role for CXCR4 in the metastasis of melanoma cells (18).Rho GTPases control the dynamics of the actin cytoskeleton during cell migration (19, 20). The activity of Rho GTPases is tightly regulated by guanine-nucleotide exchange factors (GEFs),⁴ which stimulate exchange of bound GDP by GTP, and inhibited by GTPase-activating proteins (GAPs), which promote GTP hydrolysis (21, 22), whereas guanine nucleotide dissociation inhibitors (GDIs) appear to mediate blocking of spontaneous activation (23). Therefore, cell migration is finely regulated by the balance between GEF, GAP, and GDI activities on Rho GTPases. Involvement of Rho GTPases in cancer is well documented (reviewed in Ref. 24), providing control of both cell migration and growth. RhoA and RhoC are highly expressed in colon, breast, and lung carcinoma (25, 26), whereas overexpression of RhoC in melanoma leads to enhancement of cell metastasis (27). CXCL12 activates both RhoA and Rac1 in melanoma cells, and both GTPases play key roles during invasion toward this chemokine (28, 29).Given the importance of the CXCL12-CXCR4 axis in melanoma cell invasion and metastasis, in this study we have addressed the question of whether changes in E-cadherin expression on melanoma cells might affect cell invasiveness. We show here that overexpression of E-cadherin leads to impaired melanoma cell invasion to CXCL12, and we provide mechanistic characterization accounting for the decrease in invasion.     

Engineered Protein Connectivity to Actin Mimics PDZ-dependent Recycling of G Protein-coupled Receptors but Not Its Regulation by Hrs

Many G protein-coupled receptors (GPCRs) recycle after agonist-induced endocytosis by a sequence-dependent mechanism, which is distinct from default membrane flow and remains poorly understood. Efficient recycling of the β2-adrenergic receptor (β2AR) requires a C-terminal PDZ (PSD-95/Discs Large/ZO-1) protein-binding determinant (PDZbd), an intact actin cytoskeleton, and is regulated by the endosomal protein Hrs (hepatocyte growth factor-regulated substrate). The PDZbd is thought to link receptors to actin through a series of protein interaction modules present in NHERF/EBP50 (Na⁺/H+ exchanger 3 regulatory factor/ezrin-binding phosphoprotein of 50 kDa) family and ERM (ezrin/radixin/moesin) family proteins. It is not known, however, if such actin connectivity is sufficient to recapitulate the natural features of sequence-dependent recycling. We addressed this question using a receptor fusion approach based on the sufficiency of the PDZbd to promote recycling when fused to a distinct GPCR, the δ-opioid receptor, which normally recycles inefficiently in HEK293 cells. Modular domains mediating actin connectivity promoted receptor recycling with similarly high efficiency as the PDZbd itself, and recycling promoted by all of the domains was actin-dependent. Regulation of receptor recycling by Hrs, however, was conferred only by the PDZbd and not by downstream interaction modules. These results suggest that actin connectivity is sufficient to mimic the core recycling activity of a GPCR-linked PDZbd but not its cellular regulation.G protein-coupled receptors (GPCRs)² comprise the largest family of transmembrane signaling receptors expressed in animals and transduce a wide variety of physiological and pharmacological information. While these receptors share a common 7-transmembrane-spanning topology, structural differences between individual GPCR family members confer diverse functional and regulatory properties (1-4). A fundamental mechanism of GPCR regulation involves agonist-induced endocytosis of receptors via clathrin-coated pits (4). Regulated endocytosis can have multiple functional consequences, which are determined in part by the specificity with which internalized receptors traffic via divergent downstream membrane pathways (5-7).Trafficking of internalized GPCRs to lysosomes, a major pathway traversed by the δ-opioid receptor (δOR), contributes to proteolytic down-regulation of receptor number and produces a prolonged attenuation of subsequent cellular responsiveness to agonist (8, 9). Trafficking of internalized GPCRs via a rapid recycling pathway, a major route traversed by the β2-adrenergic receptor (β2AR), restores the complement of functional receptors present on the cell surface and promotes rapid recovery of cellular signaling responsiveness (6, 10, 11). When co-expressed in the same cells, the δOR and β2AR are efficiently sorted between these divergent downstream membrane pathways, highlighting the occurrence of specific molecular sorting of GPCRs after endocytosis (12).Recycling of various integral membrane proteins can occur by default, essentially by bulk membrane flow in the absence of lysosomal sorting determinants (13). There is increasing evidence that various GPCRs, such as the β2AR, require distinct cytoplasmic determinants to recycle efficiently (14). In addition to requiring a cytoplasmic sorting determinant, sequence-dependent recycling of the β2AR differs from default recycling in its dependence on an intact actin cytoskeleton and its regulation by the conserved endosomal sorting protein Hrs (hepatocyte growth factor receptor substrate) (11, 14). Compared with the present knowledge regarding protein complexes that mediate sorting of GPCRs to lysosomes (15, 16), however, relatively little is known about the biochemical basis of sequence-directed recycling or its regulation.The β2AR-derived recycling sequence conforms to a canonical PDZ (PSD-95/Discs Large/ZO-1) protein-binding determinant (henceforth called PDZbd), and PDZ-mediated protein association(s) with this sequence appear to be primarily responsible for its endocytic sorting activity (17-20). Fusion of this sequence to the cytoplasmic tail of the δOR effectively re-routes endocytic trafficking of engineered receptors from lysosomal to recycling pathways, establishing the sufficiency of the PDZbd to function as a transplantable sorting determinant (18). The β2AR-derived PDZbd binds with relatively high specificity to the NHERF/EBP50 family of PDZ proteins (21, 22). A well-established biochemical function of NHERF/EBP50 family proteins is to associate integral membrane proteins with actin-associated cytoskeletal elements. This is achieved through a series of protein-interaction modules linking NHERF/EBP50 family proteins to ERM (ezrin-radixin-moesin) family proteins and, in turn, to actin filaments (23-26). Such indirect actin connectivity is known to mediate other effects on plasma membrane organization and function (23), however, and NHERF/EBP50 family proteins can bind to additional proteins potentially important for endocytic trafficking of receptors (23, 25). Thus it remains unclear if actin connectivity is itself sufficient to promote sequence-directed recycling of GPCRs and, if so, if such connectivity recapitulates the normal cellular regulation of sequence-dependent recycling. In the present study, we took advantage of the modular nature of protein connectivity proposed to mediate β2AR recycling (24, 26), and extended the opioid receptor fusion strategy used successfully for identifying diverse recycling sequences in GPCRs (27-29), to address these fundamental questions.Here we show that the recycling activity of the β2AR-derived PDZbd can be effectively bypassed by linking receptors to ERM family proteins in the absence of the PDZbd itself. Further, we establish that the protein connectivity network can be further simplified by fusing receptors to an interaction module that binds directly to actin filaments. We found that bypassing the PDZ-mediated interaction using either domain is sufficient to mimic the ability of the PDZbd to promote efficient, actin-dependent recycling of receptors. Hrs-dependent regulation, however, which is characteristic of sequence-dependent recycling of wild-type receptors, was recapitulated only by the fused PDZbd and not by the proposed downstream interaction modules. These results support a relatively simple architecture of protein connectivity that is sufficient to mimic the core recycling activity of the β2AR-derived PDZbd, but not its characteristic cellular regulation. Given that an increasing number of GPCRs have been shown to bind PDZ proteins that typically link directly or indirectly to cytoskeletal elements (17, 27, 30-32), the present results also suggest that actin connectivity may represent a common biochemical principle underlying sequence-dependent recycling of various GPCRs.     

Secreted Trefoil Factor 2 Activates the CXCR4 Receptor in Epithelial and Lymphocytic Cancer Cell Lines

The secreted trefoil factor family 2 (TFF2) protein contributes to the protection of the gastrointestinal mucosa from injury by strengthening and stabilizing mucin gels, stimulating epithelial restitution, and restraining the associated inflammation. Although trefoil factors have been shown to activate signaling pathways, no cell surface receptor has been directly linked to trefoil peptide signaling. Here we demonstrate the ability of TFF2 peptide to activate signaling via the CXCR4 chemokine receptor in cancer cell lines. We found that both mouse and human TFF2 proteins (at ∼0.5 μm) activate Ca²⁺ signaling in lymphoblastic Jurkat cells that could be abrogated by receptor desensitization (with SDF-1α) or pretreatment with the specific antagonist AMD3100 or an anti-CXCR4 antibody. TFF2 pretreatment of Jurkat cells decreased Ca²⁺ rise and chemotactic response to SDF-1α. In addition, the CXCR4-negative gastric epithelial cell line AGS became highly responsive to TFF2 treatment upon expression of the CXCR4 receptor. TFF2-induced activation of mitogen-activated protein kinases in gastric and pancreatic cancer cells, KATO III and AsPC-1, respectively, was also dependent on the presence of the CXCR4 receptor. Finally we demonstrate a distinct proliferative effect of TFF2 protein on an AGS gastric cancer cell line that expresses CXCR4. Overall these data identify CXCR4 as a bona fide signaling receptor for TFF2 and suggest a mechanism through which TFF2 may modulate immune and tumorigenic responses in vivo.Trefoil factor 2 (TFF2),² previously known as spasmolytic polypeptide, is a unique member of the trefoil family that is expressed primarily in gastric mucous neck cells and is up-regulated in the setting of chronic inflammation. Experimental induction of ulceration in the rat stomach leads to rapid up-regulation of TFF2 expression with high levels observed 30 min after ulceration with persistence for up to 10 days (1). TFF2 is secreted into the mucus layer of the gastrointestinal tract of mammals where it stabilizes the mucin gel layer and stimulates migration of epithelial cells (2–4), suggesting an important role in restitution and in maintenance of the integrity of the gut. Exogenous administration of recombinant TFF2, either orally or intravenously, provides mucosal protection in several rodent models of acute gastric or intestinal injury (5, 6). A TFF2^-/- knock-out mouse model has confirmed the importance of TFF2 in the protection of gastrointestinal mucosa against chronic injury (7).It is widely accepted that trefoil factors exert their biological action through a cell surface receptor. This suggestion comes from studies on binding of ¹²⁵I-labeled TFF2 that demonstrated specific binding sites in the gastric glands, intestine, and colon that could be displaced by non-radioactive TFF2 (6, 8–10). Structural studies have revealed potential binding sites for receptors for all members of the trefoil factor family (11, 12). In concordance with this hypothesis, several membrane proteins were found to interact with TFF2. First it was shown that recombinant human TFF2 (and TFF3) could bind to a 28-kDa peptide from membrane fractions of rat jejunum and two human adenocarcinoma cell lines, MCF-7 and Colony-29 (13). Later it was found that recombinant TFF3 fused with biotin selectively bound with a 50-kDa protein from the membrane of rat small intestinal cells (14). However, these 28- and 50-kDa proteins were characterized only by their molecular size without further identification. Two TFF2-binding proteins that have been characterized include a 140-kDa protein, the β subunit of the fibronectin receptor, and a 224-kDa protein called muclin (15). Another TFF2-binding protein was isolated by probing two-dimensional blots of mouse stomach with a murine TFF2 fusion protein, leading to the identification of the gastric foveolar protein blottin, a murine homolog of the human peptide TFIZ1(16). Although these three proteins have now been well characterized, none of them has been shown to mediate responses to TFF2, and no activated signaling cascades have been shown.Despite the absence of an identified cell surface receptor for TFF2, there is nevertheless clear evidence that TFF2 and TFF3 rapidly activate signal transduction pathways (17, 18). TFF3 prevents cell death via activation of the serine/threonine kinase AKT in colon cancer cell lines (19). The TFF3 protein also activates STAT3 signaling in human colorectal cancer cells, thus providing cells with invasion potential (20). TFF3 treatment leads to EGF receptor activation and β-catenin phosphorylation in HT-29 cells (21) and to transient phosphorylation of ERK1/2 in oral keratinocytes (22). With respect to TFF2, recombinant peptide enhances the migration of human bronchial epithelial cell line BEAS-2B (4). TFF2 has been shown to induce phosphorylation of c-Jun NH₂-terminal kinase (JNK) and ERK1/2. Consistent with this observation, the motogenic effect of TFF2 is significantly inhibited by antagonists of ERK kinases and protein kinase C but not by inhibitors of p38 mitogen-activated protein kinase (MAPK). It is believed that the motogenic effect of trefoil factors and of TFF2 in particular, could contribute to in vivo restitution of gastric epithelium by enhancing cell migration.Although previous studies have suggested that TFF2 functions primarily in cytoprotection, accumulating evidence now suggests that TFF2 may also play a role in the regulation of host immunity. For example, recombinant TFF2 reduces inflammation in rat and mouse models of colitis (23, 24). In addition, TFF2 was detected in rat lymphoid tissues (spleen, lymph nodes, and bone marrow) (25). Recently we and others found TFF2 mRNA expression in primary and secondary lymphopoietic organs (26, 27). These data suggest that TFF2 may play some function in the immune system. In concordance with these findings, we detected an exacerbated inflammatory response to acute injury in TFF2 knock-out animals (27, 28). These observations prompted us to look at the possible function of TFF2 in immune cells. Unexpectedly we found that TFF2 modulates Ca²⁺ and AKT signaling in lymphoblastic Jurkat cells and that these effects appear to be mediated through the CXCR4 receptor.