The Activity of the Epithelial Sodium Channels Is Regulated by Caveolin-1 via a Nedd4-2-dependent Mechanism

It has recently been shown that the epithelial Na⁺ channel (ENaC) is compartmentalized in caveolin-rich lipid rafts and that pharmacological depletion of membrane cholesterol, which disrupts lipid raft formation, decreases the activity of ENaC. Here we show, for the first time, that a signature protein of caveolae, caveolin-1 (Cav-1), down-regulates the activity and membrane surface expression of ENaC. Physical interaction between ENaC and Cav-1 was also confirmed in a coimmunoprecipitation assay. We found that the effect of Cav-1 on ENaC requires the activity of Nedd4-2, a ubiquitin protein ligase of the Nedd4 family, which is known to induce ubiquitination and internalization of ENaC. The effect of Cav-1 on ENaC requires the proline-rich motifs at the C termini of the β- and γ-subunits of ENaC, the binding motifs that mediate interaction with Nedd4-2. Taken together, our data suggest that Cav-1 inhibits the activity of ENaC by decreasing expression of ENaC at the cell membrane via a mechanism that involves the promotion of Nedd4-2-dependent internalization of the channel.Amiloride-sensitive epithelial Na⁺ channels (ENaC)³ are membrane proteins that are expressed in salt-absorptive epithelia, including the distal collecting tubules of the kidney, the mucosa of the distal colon, the respiratory epithelium, and the excretory ducts of sweat and salivary glands (1–4). Na⁺ absorption via ENaC is critical to the normal regulation of Na⁺ and fluid homeostasis and is important for maintaining blood pressure (5) and the volume of fluid in the respiratory passages (6). Increased ENaC activity has been implicated in the salt-sensitive inherited form of hypertension, Liddle''s syndrome (7), and dehydration of the surface of the airway epithelium in the pathology associated with cystic fibrosis lung disease (8).Expression of ENaC at the cell membrane surface is regulated by the E3 ubiquitin protein ligase, Nedd4-2 (neural precursor cell expressed developmentally down-regulated protein 4) (9). Interaction between the WW domains of Nedd4-2 and the proline-rich PY motifs (PPPXY) on ENaC is essential for Nedd4-2 to exert a negative effect on the channel (10, 11). This interaction leads to ubiquitination-dependent internalization of ENaC (12, 13). Several regulators of ENaC exert their effects on the channel by modulating the action of Nedd4-2. For instance, serum and glucocorticoid-dependent protein kinase (14), protein kinase B (15), and G protein-coupled receptor kinase (16) up-regulate activity of ENaC by inhibiting Nedd4-2. Although the details of cellular mechanisms that underlie internalization of ENaC remain to be elucidated, the physiological significance of Nedd4-dependent internalization of the channel has been well established. For instance, heritable mutations that delete the cytosolic termini of the β-or γ-subunit of ENaC, which contain the proline-rich motifs, are known to cause hyperactivity of ENaC in the kidney (17) and increase cell surface expression of the channel (7, 18).The plasma membranes of most cell types contain lipid raft microdomains that are enriched with glycosphingolipid and cholesterol (19), that have distinctive biophysical properties, and that selectively include or exclude signaling molecules (20). These microdomains promote clustering of an array of integral membrane proteins in the membrane leaflets (21) and may be important for organizing cascades of signaling molecules (22, 23). Processes in which raft microdomains are involved include the intracellular transport of proteins and lipids to the cell membrane (24), the endocytotic retrieval of membrane proteins (25, 26), and signal transduction (27, 28). In addition, segregation of signaling molecules within lipid rafts may facilitate cross-talk between signal transduction pathways (29), a phenomenon that may be important in ensuring rapid and efficient integration of multiple cellular signaling events (30, 31). Of particular interest is the subpopulation of lipid rafts enriched with caveolin proteins. Caveolin-1 (Cav-1), a major caveolin isoform expressed in nonmuscle cells, has been identified as being involved in diverse cellular functions, such as vesicular transport, cholesterol homeostasis, and signal transduction (32). Cav-1 also regulates the activity and membrane expression of ion channels and transporters (28).In epithelia, the majority of lipid rafts exist at the apical membrane surface (22). Pools of ENaC (33–36) and several proteins that regulate activity of ENaC, such as Nedd4 (37), protein kinase B (38), protein kinase C (39), G_o (40), and the G protein-coupled receptor kinase (41), have been identified in detergent-insoluble and cholesterol-rich membrane fractions from a variety of cell types, consistent with localization of these proteins in lipid rafts. Furthermore, detergent-free buoyant density separation of lipid rafts has revealed the presence of Cav-1 with ENaC in the lipid raft-rich membrane fraction (35). The physiological role of lipid rafts in the regulation of ENaC has been the subject of many recent investigations. Most of these studies used a pharmacological agent, methyl-β-cyclodextrin (MβCD), to promote redistribution of proteins away from the cholesterol-enriched membrane domains. The results were, however, inconclusive. In some studies, MβCD treatment was found to inhibit open probability (42) or cell surface expression of ENaC (35), whereas others found no direct effect of MβCD on the channel (33, 43).Despite a number of studies into the role of lipid rafts on the regulation of ENaC, little is known about the physiological relevance of caveolins to the function of this ion channel. In the present study, we use gene interference and gene expression techniques to determine the role of Cav-1 in the regulation of ENaC activity. We provide evidence of the association of Cav-1 with ENaC and evidence that Cav-1 negatively regulates both activity and abundance of ENaC at the surface of epithelial cells. Importantly, we demonstrate, for the first time, that the mechanism by which Cav-1 regulates activity of ENaC involves the E3 ubiquitin protein ligase, Nedd4-2.     

ATR and H2AX Cooperate in Maintaining Genome Stability under Replication Stress

Chromosomal abnormalities are frequently caused by problems encountered during DNA replication. Although the ATR-Chk1 pathway has previously been implicated in preventing the collapse of stalled replication forks into double-strand breaks (DSB), the importance of the response to fork collapse in ATR-deficient cells has not been well characterized. Herein, we demonstrate that, upon stalled replication, ATR deficiency leads to the phosphorylation of H2AX by ATM and DNA-PKcs and to the focal accumulation of Rad51, a marker of homologous recombination and fork restart. Because H2AX has been shown to play a facilitative role in homologous recombination, we hypothesized that H2AX participates in Rad51-mediated suppression of DSBs generated in the absence of ATR. Consistent with this model, increased Rad51 focal accumulation in ATR-deficient cells is largely dependent on H2AX, and dual deficiencies in ATR and H2AX lead to synergistic increases in chromatid breaks and translocations. Importantly, the ATM and DNA-PK phosphorylation site on H2AX (Ser¹³⁹) is required for genome stabilization in the absence of ATR; therefore, phosphorylation of H2AX by ATM and DNA-PKcs plays a pivotal role in suppressing DSBs during DNA synthesis in instances of ATR pathway failure. These results imply that ATR-dependent fork stabilization and H2AX/ATM/DNA-PKcs-dependent restart pathways cooperatively suppress double-strand breaks as a layered response network when replication stalls.Genome maintenance prevents mutations that lead to cancer and age-related diseases. A major challenge in preserving genome integrity occurs in the simple act of DNA replication, in which failures at numerous levels can occur. Besides the mis-incorporation of nucleotides, it is during this phase of the cell cycle that the relatively stable double-stranded nature of DNA is temporarily suspended at the replication fork, a structure that is susceptible to collapse into DSBs.² Replication fork stability is maintained by a variety of mechanisms, including activation of the ATR-dependent checkpoint pathway.The ATR pathway is activated upon the generation and recognition of extended stretches of single-stranded DNA at stalled replication forks (1-4). Genome maintenance functions for ATR and orthologs in yeast were first indicated by increased chromatid breaks in ATR^-/- cultured cells (5) and by the “cut” phenotype observed in Mec1 (Saccharomyces cerevisiae) and Rad3 (Schizosaccharomyces pombe) mutants (6-9). Importantly, subsequent studies in S. cerevisiae demonstrated that mutation of Mec1 or the downstream checkpoint kinase Rad53 led to increased chromosome breaks at regions of the genome that are inherently difficult to replicate (10), and a decreased ability to reinitiate replication fork progression following DNA damage or deoxyribonucleotide depletion (11-14).In vertebrates, similar replication fork stabilizing functions have been demonstrated for ATR and the downstream protein kinase Chk1 (15-20). Several possible mechanisms have been put forward to explain how ATR-Chk1 and orthologous pathways in yeast maintain replication fork stability, including maintenance of replicative polymerases (α, δ, and ε) at forks (17, 21), regulation of branch migrating helicases, such as Blm (22-25), and regulation of homologous recombination, either positively or negatively (26-29).Consistent with the role of the ATR-dependent checkpoint in replication fork stability, common fragile sites, located in late-replicating regions of the genome, are significantly more unstable (5-10-fold) in the absence of ATR or Chk1 (19, 20). Because these sites are favored regions of instability in oncogene-transformed cells and preneoplastic lesions (30, 31), it is possible that the increased tumor incidence observed in ATR haploinsufficient mice (5, 32) may be related to subtle increases in genomic instability. Together, these studies indicate that maintenance of replication fork stability may contribute to tumor suppression.It is important to note that prevention of fork collapse represents an early response to problems occurring during DNA replication. In the event of fork collapse into DSBs, homologous recombination (HR) has also been demonstrated to play a key role in genome stability during S phase by catalyzing recombination between sister chromatids as a means to re-establish replication forks (33). Importantly, a facilitator of homologous recombination, H2AX, has been shown to be phosphorylated under conditions that cause replication fork collapse (18, 34).Phosphorylation of H2AX occurs predominantly upon DSB formation (34-38) and has been reported to require ATM, DNA-PKcs, or ATR, depending on the context (37-42). Although H2AX is not essential for HR, studies have demonstrated that H2AX mutation leads to deficiencies in HR (43, 44), and suppresses events associated with homologous recombination, such as the focal accumulation of Rad51, BRCA1, BRCA2, ubiquitinated-FANCD2, and Ubc13-mediated chromatin ubiquitination (43, 45-51). Therefore, through its contribution to HR, it is possible that H2AX plays an important role in replication fork stability as part of a salvage pathway to reinitiate replication following collapse.If ATR prevents the collapse of stalled replication forks into DSBs, and H2AX facilitates HR-mediated restart, the combined deficiency in ATR and H2AX would be expected to dramatically enhance the accumulation of DSBs upon replication fork stalling. Herein, we utilize both partial and complete elimination of ATR and H2AX to demonstrate that these genes work cooperatively in non-redundant pathways to suppress DSBs during S phase. As discussed, these studies imply that the various components of replication fork protection and regeneration cooperate to maintain replication fork stability. Given the large number of genes involved in each of these processes, it is possible that combined deficiencies in these pathways may be relatively frequent in humans and may synergistically influence the onset of age-related diseases and cancer.     

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.     

Interaction of Mint2 with TrkA Is Involved in Regulation of Nerve Growth Factor-induced Neurite Outgrowth

TrkA receptor signaling is essential for nerve growth factor (NGF)-induced survival and differentiation of sensory neurons. To identify possible effectors or regulators of TrkA signaling, yeast two-hybrid screening was performed using the intracellular domain of TrkA as bait. We identified muc18-1-interacting protein 2 (Mint2) as a novel TrkA-binding protein and found that the phosphotyrosine binding domain of Mint2 interacted with TrkA in a phosphorylation- and ligand-independent fashion. Coimmunoprecipitation assays showed that endogenous TrkA interacted with Mint2 in rat tissue homogenates, and immunohistochemical evidence revealed that Mint2 and TrkA colocalized in rat dorsal root ganglion neurons. Furthermore, Mint2 overexpression inhibited NGF-induced neurite outgrowth in both PC12 and cultured dorsal root ganglion neurons, whereas inhibition of Mint2 expression by RNA interference facilitated NGF-induced neurite outgrowth. Moreover, Mint2 was found to promote the retention of TrkA in the Golgi apparatus and inhibit its surface sorting. Taken together, our data provide evidence that Mint2 is a novel TrkA-regulating protein that affects NGF-induced neurite outgrowth, possibly through a mechanism involving retention of TrkA in the Golgi apparatus.The neurotrophin family member nerve growth factor (NGF)³ is essential for proper development, patterning, and maintenance of nervous systems (1, 2). NGF has two known receptors; TrkA, a single-pass transmembrane receptor-tyrosine kinase that binds selectively to NGF, and p75, a transmembrane glycoprotein that binds all members of the neurotrophin family (3, 4). NGF binding activates the kinase domain of TrkA, leading to autophosphorylation (5). The resulting phosphotyrosines become docking sites for adaptor proteins involved in signal transduction pathways that lead to the activation of Ras, Rac, phosphatidylinositol 3-kinase, phospholipase Cγ, and other effectors (2, 6). Many of these TrkA-interacting adaptor proteins have been identified and include, Grb2, APS, SH2B, fibroblast growth factor receptor substrate 2 (FRS-2), Shc, and human tumor imaginal disc 1 (TID1) (7-10). The identification of these binding partners has contributed greatly to our understanding of the mechanisms underlying the functional diversity of NGF-TrkA signaling.Studies have indicated that the transmission of NGF signaling in neurons involves retrograde transport of NGF-TrkA complexes from the neurite tip to the cell body (11-14). TrkA associates with components of cytoplasmic dynein, and it is thought that vesicular trafficking of neurotrophins occurs via direct interaction of Trk receptors with the dynein motor machinery (14). Furthermore, the atypical protein kinase C-interacting protein, p62, associates with TrkA and plays a novel role in connecting receptor signals with the endosomal signaling network required for mediating TrkA-induced differentiation (15). Recently, the membrane-trafficking protein Pincher has been found to mediate macroendocytosis underlying retrograde signaling by TrkA (16). Despite the progress made to date in understanding Trk complex internalization and trafficking, the mechanisms remain poorly understood.Mint2 (muc18-1-interacting protein 2) belongs to the Mint protein family, which consists of three members, Mint1, Mint2, and Mint3. Mint proteins were first identified as interacting proteins of the synaptic vesicle-docking protein Munc18-1 (17, 18). Mint1 is also sometimes referred to as mLIN-10, as it is the mammalian orthologue of the Caenorhabditis elegans LIN-10 (19). Additionally, Mint1, Mint2, and Mint3 are also referred to as X11α or X11, X11β or X11L (X11-like), and X11γ or X11L2 (X11-like 2), respectively (20). All Mint proteins contain a conserved central phosphotyrosine binding (PTB) domain and two contiguous C-terminal PDZ domains (repeated sequences in the brain-specific protein PSD-95, the Drosophila septate junction protein Discs large, and the epithelial tight junction protein ZO-1) (17, 18, 21). Mint1 and Mint2 are expressed only in neuronal tissue (17), whereas Mint3 is ubiquitously expressed (18). Although the function of Mints proteins is not fully clear, their interactions with the docking and exocytosis factors Mun18 -1 and CASK, ADP-ribosylation factor (Arf) GTPases involved in vesicle budding (22), and other synaptic adaptor proteins, such as neurabin-II/spinophilin (23), tamalin (24), and kalirin-7 (25), all suggest possible roles for Mints in synaptic vesicle docking and exocytosis. Mint proteins have also been implicated in the trafficking and/or processing of β-amyloid precursor protein (β-APP). Through their PTB domains, all three Mints bind to a motif within the cytoplasmic domain of β-APP (21, 26-29), and Mint1 and Mint2 can stabilize β-APP, affect β-APP processing, and inhibit the production and secretion of Aβ (28, 30-32). Although the mechanisms by which Mints inhibit β-APP processing are not yet well known, Mints and their binding partners have emerged as potential therapeutic targets for the treatment of Alzheimer disease.To uncover new TrkA-interacting factors and gain insight into the mechanisms that guide TrkA intracellular trafficking and other aspects of TrkA signaling, we conducted a yeast two-hybrid screen of a brain cDNA library using the intracellular domain of TrkA as bait. The screen identified several candidate TrkA-interacting proteins, one of which was Mint2. Follow-up binding assays showed that the PTB domain of Mint2 alone was necessary and sufficient for mediating the interaction with TrkA. Endogenous Mint2 was also coimmunoprecipitated and colocalized with TrkA in rat DRG tissue. Overexpression and knockdown studies showed that Mint2 could significantly inhibit NGF-induced neurite outgrowth in both TrkA-expressing PC12 cells and DRG neurons. Moreover, Mint2 was found to induce the retention of TrkA in the Golgi apparatus and inhibit its surface sorting. Our results suggest that Mint2 is a novel regulator of TrkA receptor signaling.     

TRAM1 Participates in Human Cytomegalovirus US2- and US11-mediated Dislocation of an Endoplasmic Reticulum Membrane Glycoprotein

The human cytomegalovirus proteins US2 and US11 have co-opted endoplasmic reticulum (ER) quality control to facilitate the destruction of major histocompatibility complex class I heavy chains. The class I heavy chains are dislocated from the ER to the cytosol, where they are deglycosylated and subsequently degraded by the proteasome. We examined the role of TRAM1 (translocating chain-associated membrane protein-1) in the dislocation of class I molecules using US2- and US11-expressing cells. TRAM1 is an ER protein initially characterized for its role in processing nascent polypeptides. Co-immunoprecipitation studies demonstrated that TRAM1 can complex with the wild type US2 and US11 proteins as well as deglycosylated and polyubiquitinated class I degradation intermediates. In studies using US2- and US11-TRAM1 knockdown cells, we observed an increase in levels of class I heavy chains. Strikingly, increased levels of glycosylated heavy chains were observed in TRAM1 knockdown cells when compared with control cells in a pulse-chase experiment. In fact, US11-mediated class I dislocation was more sensitive to the lack of TRAM1 than US2. These results provide further evidence that these viral proteins may utilize distinct complexes to facilitate class I dislocation. For example, US11-mediated class I heavy chain degradation requires Derlin-1 and SEL1L, whereas signal peptide peptidase is critical for US2-induced class I destabilization. In addition, TRAM1 can complex with the dislocation factors Derlin-1 and signal peptide peptidase. Collectively, the data support a model in which TRAM1 functions as a cofactor to promote efficient US2- and US11-dependent dislocation of major histocompatibility complex class I heavy chains.HCMV² can down-regulate cell surface expression of the immunologically important molecule major histocompatibility complex class I to avoid immune detection by cytotoxic T cells (1, 2). More specifically, the HCMV US2 and US11 gene products alone can target the ER-localized major histocompatibility complex class I heavy chains for extraction across the ER membrane by a process referred to as dislocation or retrograde translocation. The N-linked glycan is then removed upon exposure to the cytosol by N-glycanase (3), followed by proteasomal destruction (4, 5). The HCMV US2 and US11 proteins utilize the ER quality control process to eliminate class I heavy cells in a similar manner as misfolded or damaged ER proteins (e.g. genetic mutants of α₁-antitrypsin (6) and the cystic fibrosis transmembrane conductance regulator protein (7)) are targeted for degradation (8). Hence, analysis of US2- and US11-mediated destruction of class I heavy chains provides an excellent system to delineate viral protein function as well as the ER quality control process.ER and cytosolic proteins are required for US2- and US11-mediated dislocation/degradation of class I heavy chains. Some of these proteins have also been identified in the processing of aberrant ER polypeptides. The ER chaperones calnexin, calreticulin, and BiP have been implicated in US2-mediated class I destruction (9) as well as in the removal of some misfolded ER proteins (10). The ubiquitination machinery also participates in the extraction of class I heavy chains as ubiquitinated heavy chains are observed prior to dislocation (11, 12). For misfolded ER degradation substrates, ubiquitin conjugation enzymes (e.g. Ubc6p and Ubc7p/Cue1p) and ubiquitin ligases Hrd1p/Der3p, Doa10p, and Ubc1p have been implicated in the dislocation reaction (8). Interestingly, the ER membrane protein Derlin-1 along with SEL1L are involved in US11-mediated class I heavy chain degradation (13-15), whereas SPP is critical for US2-induced class I destabilization (16). The ubiquitinated substrates are dislocated by the AAA-ATPase complex composed of p97-Ufd1-Npl4 (17) while docked to the ER through its interaction with VIMP (14) followed by proteasome destruction. The inhibition of the proteasome causes the accumulation of deglycosylated class I heavy chain intermediate in US2 and US11 cells, allowing the dislocation and degradation reactions to be studied as separate processes (4, 5).Despite the identification of some cellular proteins that assist US2- and US11-mediated class I dislocation, the dislocation pore and accessory factors that mediate the efficient extraction of class I through the bilayer have yet to be completely defined. The current study explores the role of TRAM1 (translocating chain-associated membrane protein-1) in US2- and US11-mediated class I dislocation. TRAM1 is an ER-resident multispanning membrane protein that can mediate the lateral movement of select signal peptides and transmembrane segments from the translocon into the membrane bilayer (18), a property that makes it uniquely qualified to participate in the dislocation of a membrane protein. TRAM1 has been cross-linked to signal peptides as well as transmembrane domains of nascent polypeptides during the early stages of protein processing (19-25). Interestingly, unlike the Sec61 complex and the signal recognition particle receptor, TRAM1 is not essential for the translocation of all membrane proteins into the ER (20, 21). Hence, TRAM1 may utilize its ability to engage hydrophobic domains to assist in the efficient dislocation of membrane proteins. In fact, association and TRAM1 knockdown studies demonstrate that TRAM1 participates in US2- and US11-mediated dislocation of class I heavy chains. Collectively, our data suggest for the first time that TRAM1 plays a role in the dislocation of a membrane glycoprotein.     

Crystal Structure of the N-terminal Domain of Anaphase-promoting Complex Subunit 7

Anaphase-promoting complex or cyclosome (APC/C) is an unusual E3 ubiquitin ligase and an essential protein that controls mitotic progression. APC/C includes at least 13 subunits, but no structure has been determined for any tetratricopeptide repeat (TPR)-containing subunit (Apc3 and -6-8) in the TPR subcomplex of APC/C. Apc7 is a TPR-containing subunit that exists only in vertebrate APC/C. Here we report the crystal structure of quad mutant of nApc7 (N-terminal fragment, residues 1-147) of human Apc7 at a resolution of 2.5 Å. The structure of nApc7 adopts a TPR-like motif and has a unique dimerization interface, although the protein does not contain the conserved TPR sequence. Based on the structure of nApc7, in addition to previous experimental findings, we proposed a putative homodimeric structure for full-length Apc7. This model suggests that TPR-containing subunits self-associate and bind to adaptors and substrates via an IR peptide in TPR-containing subunits of APC/C.Anaphase-promoting complex/cyclosome (APC/C)² is an E3 ubiquitin ligase that controls mitotic progression (1). APC/C is an ∼1.7-MDa protein complex that is composed of at least 13 subunits, and it contains a cullin homolog (Apc2), a ring-H2 finger domain (Apc11), and a tetratricopeptide repeat (TPR)-containing subunit (TPR subunit; Apc3 and -6-8) (2). Most TPR subunits are essential and evolutionarily conserved in eukaryotes (3).APC/C requires two adaptors that contain a C-terminal WD40 domain, Cdc20 and Cdh1, to recruit and select various substrates at different stages of the cell cycle. Moreover, both adaptors and specific APC/C subunits contribute to substrate recognition (4).APC/C specifically ubiquitinates cell cycle regulatory proteins that contain destruction (D) or KEN box motifs (5-7), which target them for destruction by the 26 S proteosome (8). During the cell cycle, APC/C mediates the metaphase-anaphase transition by ubiquitinating and degrading securin, a separase inhibitor, which participates in the degradation of chromatic cohesion complexes and ubiquitinates B-type cyclin, thereby accelerating transition from the late mitotic phase to G₁ (9). In addition to its primary role in cell cycle regulation, APC/C participates in postmitotic processes, such as regulation of synaptic size and axon growth (10, 11).To assess the mechanism that underlies cell cycle regulation by APC/C and the various roles of its subunits, we need to understand how APC/C is organized into higher order structures and the manner in which the subunits assemble. Although little is known regarding the crystal structures of APC/C components, three-dimensional models of APC/C have recently been obtained by cryo-negative staining EM in human, Xenopus laevis, Saccharomyces cerevisiae, and Schizosaccharomyces pombe (12-15). Several studies have indicated that APC/C assumes an asymmetric triangular shape that is composed of an outer shell and a cavity that extends through its center (12, 14). Furthermore, APC/C includes a catalytic subcomplex (Doc1/Apc10, Apc11, and Apc2), a structural complex (Apc1, Apc4, and Apc5), and a TPR subcomplex (TPR-containing subunits and nonessential subunits) (16).A TPR unit consists of a 34-residue repeat motif that adopts a helix-turn-helix conformation, which is associated with protein-protein interactions (17). Multiple copies of TPR-containing subunits are organized into the TPR subcomplex within APC/C, and this subcomplex is functionally important for the recruitment of adaptors and substrates (18). In fact, adaptors (Cdc20 and Cdh1) and Doc1/Apc10 bind to the C-terminal domain of the TPR-containing subunits Apc3 and Apc7 via the IR peptide tail sequence (7, 16, 19). It is unknown, however, how TPR-containing subunits form homo- and heterosubunit complexes, although studies have demonstrated that TPR-containing subunits self-associate in vivo and in vitro (15) and that they interact with other TPR-containing subunits (20).Apc7 is found only in vertebrate APC/C and is estimated to contain 9-15 TPR motifs, similar to other TPR-containing subunits (9). Apc7 is considered to be a molecular descendant of the same ancestral protein that gave rise to Apc3. Furthermore, the N-terminal domain of Apc7 has been reported to contain cell cycle-regulated phosphorylation sites (21), and the C-terminal TPR domain of Apc7 interacts with Cdh1 and Cdc20 (19). In Drosophila APC/C, the homolog of vertebrate Apc7 participates in synergistic genetic interactions with other TPR-containing subunits (22).The function of Apc7 within vertebrate APC/C, however, is poorly understood. Moreover, although the C-terminal regions of Apc3 and Apc7 include a tandem of nine TPR motifs, the N-terminal domains of human Apc3 and Apc7 share little homology with the canonical TPR sequence. Therefore, the N-terminal domain of human Apc7 is expected to have a significant function in vertebrate APC/C.In this study, we determined the crystal structure of the N-terminal fragment of human Apc7 (residues 1-147, denoted nApc7), and the homodimeric self-association of nApc7 structure led us to insights into mechanisms of vertebrate APC/C.     

Energetic Requirements for Processive Elongation of Actin Filaments by FH1FH2-formins

Formin-homology (FH) 2 domains from formin proteins associate processively with the barbed ends of actin filaments through many rounds of actin subunit addition before dissociating completely. Interaction of the actin monomer-binding protein profilin with the FH1 domain speeds processive barbed end elongation by FH2 domains. In this study, we examined the energetic requirements for fast processive elongation. In contrast to previous proposals, direct microscopic observations of single molecules of the formin Bni1p from Saccharomyces cerevisiae labeled with quantum dots showed that profilin is not required for formin-mediated processive elongation of growing barbed ends. ATP-actin subunits polymerized by Bni1p and profilin release the γ-phosphate of ATP on average >2.5 min after becoming incorporated into filaments. Therefore, the release of γ-phosphate from actin does not drive processive elongation. We compared experimentally observed rates of processive elongation by a number of different FH2 domains to kinetic computer simulations and found that actin subunit addition alone likely provides the energy for fast processive elongation of filaments mediated by FH1FH2-formin and profilin. We also studied the role of FH2 structure in processive elongation. We found that the flexible linker joining the two halves of the FH2 dimer has a strong influence on dissociation of formins from barbed ends but only a weak effect on elongation rates. Because formins are most vulnerable to dissociation during translocation along the growing barbed end, we propose that the flexible linker influences the lifetime of this translocative state.Formins are multidomain proteins that assemble unbranched actin filament structures for diverse processes in eukaryotic cells (reviewed in Ref. 1). Formins stimulate nucleation of actin filaments and, in the presence of the actin monomer-binding protein profilin, speed elongation of the barbed ends of filaments (2-6). The ability of formins to influence elongation depends on the ability of single formin molecules to remain bound to a growing barbed end through multiple rounds of actin subunit addition (7, 8). To stay associated during subunit addition, a formin molecule must translocate processively on the barbed end as each actin subunit is added (1, 9-12). This processive elongation of a barbed end by a formin is terminated when the formin dissociates stochastically from the growing end during translocation (4, 10).The formin-homology (FH)² 1 and 2 domains are the best conserved domains of formin proteins (2, 13, 14). The FH2 domain is the signature domain of formins, and in many cases, is sufficient for both nucleation and processive elongation of barbed ends (2-4, 7, 15). Head-to-tail homodimers of FH2 domains (12, 16) encircle the barbed ends of actin filaments (9). In vitro, association of barbed ends with FH2 domains slows elongation by limiting addition of free actin monomers. This “gating” behavior is usually explained by a rapid equilibrium of the FH2-associated end between an open state competent for actin monomer association and a closed state that blocks monomer binding (4, 9, 17).Proline-rich FH1 domains located N-terminal to FH2 domains are required for profilin to stimulate formin-mediated elongation. Individual tracks of polyproline in FH1 domains bind 1:1 complexes of profilin-actin and transfer the actin directly to the FH2-associated barbed end to increase processive elongation rates (4-6, 8, 10, 17).Rates of elongation and dissociation from growing barbed ends differ widely for FH1FH2 fragments from different formin homologs (4). We understand few aspects of FH1FH2 domains that influence gating, elongation or dissociation. In this study, we examined the source of energy for formin-mediated processive elongation, and the influence of FH2 structure on elongation and dissociation from growing ends. In contrast to previous proposals (6, 18), we found that fast processive elongation mediated by FH1FH2-formins is not driven by energy from the release of the γ-phosphate from ATP-actin filaments. Instead, the data show that the binding of an actin subunit to the barbed end provides the energy for processive elongation. We found that in similar polymerizing conditions, different natural FH2 domains dissociate from growing barbed ends at substantially different rates. We further observed that the length of the flexible linker between the subunits of a FH2 dimer influences dissociation much more than elongation.     

Acetylation/Deacetylation Modulates the Stability of DNA Replication Licensing Factor Cdt1

Proper expression of the replication licensing factor Cdt1 is primarily regulated post-translationally by ubiquitylation and proteasome degradation. In a screen to identify novel non-histone targets of histone deacetylases (HDACs), we found Cdt1 as a binding partner for HDAC11. Cdt1 associates specifically and directly with HDAC11. We show that Cdt1 undergoes acetylation and is reversibly deacetylated by HDAC11. In vitro, Cdt1 can be acetylated at its N terminus by the lysine acetyltransferases KAT2B and KAT3B. Acetylation protects Cdt1 from ubiquitylation and subsequent proteasomal degradation. These results extend the list of non-histone acetylated proteins to include a critical DNA replication factor and provide an additional level of complexity to the regulation of Cdt1.To maintain genomic integrity, DNA replication must be tightly controlled to ensure that each portion of the genome replicates once and only once per cell cycle (reviewed in Ref. 1). Replication licensing begins by the formation of the prereplication complex at multiple potential origins of replication. This is established sequentially, with the origin recognition complex (ORC)² proteins binding first, followed by the recruitment of Cdc6 and Cdt1, which in turn recruit the MCM2–7 proteins. MCM proteins act as the replicative helicase. The licensed replication origins are activated by cyclin-dependent kinases at the start of S phase. Licensing occurs throughout the cell cycle once S phase is complete.Cdt1 levels fluctuate throughout the cell cycle. It is destabilized at G₁/S transition, and then levels begin to climb again upon S phase completion. To prevent licensing at inappropriate times, two separate processes regulate the inactivation or destruction of Cdt1. First, geminin negatively regulates Cdt1 function by prevention of the association of Cdt1 with MCM2–7 via steric hindrance (2). Interestingly, geminin also positively regulates Cdt1 by preventing its ubiquitylation, perhaps by prevention of its interaction with an E3 ligase. This allows Cdt1 to accumulate in G₂ and M phases, to ensure adequate pools of Cdt1 to license the next cycle of replication (3). The ratio of geminin to Cdt1 likely determines whether geminin positively or negatively regulates Cdt1 (4). Second, Cdt1 is targeted for proteolysis by two distinct ubiquitin E3 ligases: the SCF-Skp2 complex and the DDB1-Cul4 complex (5). Phosphorylation by cyclin A/Cdk2 promotes interaction of Cdt1 with Skp2, leading to Cdt1 degradation during S phase (6–8). In addition, DDB1-Cul4 utilizes proliferating cell nuclear antigen as a binding platform to contact Cdt1, targeting the destruction of Cdt1 in S phase or following DNA damage (9, 10). Ubiquitylation by either of these E3 ligases promotes degradation of Cdt1 by the proteasome.Ubiquitylation occurs primarily (but not exclusively) on the ε-amino group of lysine residues. Another prominent post-translational modification that occurs on that residue is acetylation. Acetylation and, correspondingly, deacetylation can modulate the function and activity of a variety of proteins (see Ref. 11 for review). Here, we report that Cdt1 physically interacts with HDAC11, a class IV histone deacetylase (12, 13), as well as with several lysine acetyltransferases (KATs). We show that Cdt1 is an acetylated protein and further show that acetylation protects Cdt1 from ubiquitylation and subsequent proteasomal degradation. This study uncovers yet another layer of complexity to the regulation of the critical licensing factor Cdt1.     

Interdomain Flexibility in Full-length Matrix Metalloproteinase-1 (MMP-1)

The presence of extensive reciprocal conformational freedom between the catalytic and the hemopexin-like domains of full-length matrix metalloproteinase-1 (MMP-1) is demonstrated by NMR and small angle x-ray scattering experiments. This finding is discussed in relation to the essentiality of the hemopexin-like domain for the collagenolytic activity of MMP-1. The conformational freedom experienced by the present system, having the shortest linker between the two domains, when compared with similar findings on MMP-12 and MMP-9 having longer and the longest linker within the family, respectively, suggests this type of conformational freedom to be a general property of all MMPs.Matrix metalloproteinases (MMP)² are extracellular hydrolytic enzymes involved in a variety of processes including connective tissue cleavage and remodeling (1–3). All 23 members of the family are able to cleave simple peptides derived from connective tissue components such as collagen, gelatin, elastin, etc. A subset of MMPs is able to hydrolyze more resistant polymeric substrates, such as cross-linked elastin, and partially degraded collagen forms, such as gelatin and type IV collagens (4). Intact triple helical type I–III collagen is only attacked by collagenases MMP-1, MMP-8, and MMP-13 and by MMP-2 and MMP-14 (5–12). Although the detailed mechanism of cleavage of single chain peptides by MMP has been largely elucidated (13–19), little is known about the process of hydrolysis of triple helical collagen. In fact, triple helical collagen cannot be accommodated in the substrate-binding groove of the catalytic site of MMPs (9).All MMPs (but MMP-7) in their active form are constituted by a catalytic domain (CAT) and a hemopexin-like domain (HPX) (20–22). The CAT domain contains two zinc ions and one to three calcium ions. One zinc ion is at the catalytic site and is responsible for the activity, whereas the other metal ions have structural roles. The isolated CAT domains retain full catalytic activity toward simple peptides and single chain polymeric substrates such as elastin, whereas hydrolysis of triple helical collagen also requires the presence of the HPX domain (9, 23–25). It has been shown that the isolated CAT domain regains a small fraction of the activity of the full-length (FL) protein when high amounts of either inactivated full-length proteins or isolated HPX domains are added to the assay solution (9). Finally, it has been shown that the presence of the HPX domain alone alters the CD spectrum of triple helical collagen in a way that suggests its partial unwinding (26, 27). It is tempting to speculate that full-length collagenases attack collagen by first locally unwinding the triple helical structure with the help of the HPX domain and then cleaving the resulting, exposed, single filaments (9, 28).Until 2007, three-dimensional structures of full-length MMPs had been reported only for collagenase MMP-1 (29–31) and gelatinase MMP-2 (32). The structures of the two proteins are very similar and show a compact arrangement of the two domains, which are connected by a short linker (14 and 20 amino acids, respectively). It is difficult to envisage that rigid and compact molecules of this type can interact with triple helical collagen in a way that can lead to first unwinding and then cleavage of individual filaments. It has been recently suggested that such concerted action could occur much more easily if the two domains could enjoy at least a partial conformational independence (9). Slight differences in the reciprocal orientation of the CAT and HPX domains of MMP-1 in the presence (29) and absence (30, 31) of the prodomain were indeed taken as a hint that the two domains could experience relative mobility (29).Two recent solution studies have shown that conformational independence is indeed occurring in gelatinase MMP-9 (33) and elastase MMP-12 (34), whereas the x-ray structure of the latter (34) is only slightly less compact than those of MMP-1 (29–31) and MMP-2 (32). Among MMPs, MMP-9 features an exceptionally long linker (68 amino acid) (33, 35), which in fact constitutes a small domain by itself (the O-glycosylated domain) (33), and therefore, this inspiring observation can hardly be taken as evidence that conformational freedom is a general characteristic of the two-domain MMPs. MMP-12 features a much more normal 16-amino acid linker, thereby making more probable a general functional role for this conformational freedom (34). However, both MMP-9 and MMP-12 retain their full catalytic activity against their substrates even when deprived of the HPX domain (9). Therefore, the question remains of whether conformational freedom is also a required characteristic for those MMPs that are only active as full-length proteins, i.e. collagenases. Interestingly, the three collagenases (MMP-1, MMP-8, and MMP-13) have the shortest linker (14 amino acids) among all MMPs. Demonstrating or negating the presence of conformational freedom in one of these collagenases would therefore constitute a significant step forward to formulate mechanistic hypotheses on their collagenolytic activity.Our recent studies on MMP-12 in solution (34) have shown that a combination of NMR relaxation studies and small angle x-ray scattering (SAXS) is enough to show the presence and the extent of the relative conformational freedom of the two domains of MMPs. Here we apply the same strategy to full-length MMP-1 and show that sizable conformational freedom is indeed experienced even by this prototypical collagenase, although somewhat less pronounced than that observed for MMP-12.     

Noonan Syndrome/Leukemia-associated Gain-of-function Mutations in SHP-2 Phosphatase (PTPN11) Enhance Cell Migration and Angiogenesis

Mutations in SHP-2 phosphatase (PTPN11) that cause hyperactivation of its catalytic activity have been identified in Noonan syndrome and various childhood leukemias. Recent studies suggest that the gain-of-function (GOF) mutations of SHP-2 play a causal role in the pathogenesis of these diseases. However, the molecular mechanisms by which GOF mutations of SHP-2 induce these phenotypes are not fully understood. Here, we show that GOF mutations in SHP-2, such as E76K and D61G, drastically increase spreading and migration of various cell types, including hematopoietic cells, endothelial cells, and fibroblasts. More importantly, in vivo angiogenesis in SHP-2 D61G knock-in mice is also enhanced. Mechanistic studies suggest that the increased cell migration is attributed to the enhanced β1 integrin outside-in signaling. In response to β1 integrin cross-linking or fibronectin stimulation, activation of ERK and Akt kinases is greatly increased by SHP-2 GOF mutations. Also, integrin-induced activation of RhoA and Rac1 GTPases is elevated. Interestingly, mutant cells with the SHP-2 GOF mutation (D61G) are more sensitive than wild-type cells to the suppression of cell motility by inhibition of these pathways. Collectively, these studies reaffirm the positive role of SHP-2 phosphatase in cell motility and suggest a new mechanism by which SHP-2 GOF mutations contribute to diseases.SHP-2, a multifunctional SH2 domain-containing protein-tyrosine phosphatase implicated in diverse cell signaling processes (1–3), plays a critical role in cellular function. Homozygous deletion of Exon 2 (4) or Exon 3 (5) of the SHP-2 gene (PTPN11) in mice leads to early embryonic lethality prior to and at midgestation, respectively. SHP-2 null mutant mice die much earlier, at peri-implantation (4). Exon 3 deletion mutation of SHP-2 blocks hematopoietic potential of embryonic stem cells both in vitro and in vivo (6–8), whereas SHP-2 null mutation causes inner cell mass death and diminished trophoblast stem cell survival (4). Recent studies on SHP-2 conditional knock-out or tissue-specific knock-out mice have further revealed an array of important functions of this phosphatase in various physiological processes (9–12). The phenotypes demonstrated by loss of SHP-2 function are apparently attributed to the role of SHP-2 in the cell signaling pathways induced by growth factors/cytokines. SHP-2 generally promotes signal transmission in growth factor/cytokine signaling in both catalytic-dependent and -independent fashion (1–3). The positive role of SHP-2 in the intracellular signaling processes, in particular, the ERK³ and PI3K/Akt kinase pathways, has been well established, although the underlying mechanism remains elusive, in particular, the signaling function of the catalytic activity of SHP-2 in these pathways is poorly understood.In addition to the role of SHP-2 in cell proliferation and differentiation, the phenotypes induced by loss of SHP-2 function may be associated with its role in cell migration. Indeed, dominant negative SHP-2 disrupts Xenopus gastrulation, causing tail truncations (13, 14). Targeted Exon 3 deletion mutation in SHP-2 results in decreased cell spreading, migration (15, 16), and impaired limb development in the chimeric mice (7). The role of SHP-2 in cell adhesion and migration has also been demonstrated by catalytically inactive mutant SHP-2-overexpressing cells (17–20). The molecular mechanisms by which SHP-2 regulates these cellular processes, however, have not been well defined. For example, the role of SHP-2 in the activation of the Rho family small GTPases that is critical for cell motility is still controversial. Both positive (19, 21, 22) and negative roles (18, 23) for SHP-2 in this context have been reported. Part of the reason for this discrepancy might be due to the difference in the cell models used. Catalytically inactive mutant SHP-2 was often used to determine the role of SHP-2 in cell signaling. In the catalytically inactive mutant SHP-2-overexpressing cells, the catalytic activity of endogenous SHP-2 is inhibited. However, as SHP-2 also functions independent of its catalytic activity, overexpression of catalytically deficient SHP-2 may also increase its scaffolding function, generating complex effects.The critical role of SHP-2 in cellular function is further underscored by the identification of SHP-2 mutations in human diseases. Genetic lesions in PTPN11 that cause hyperactivation of SHP-2 catalytic activity have been identified in the developmental disorder Noonan syndrome (24) and various childhood leukemias, including juvenile myelomonocytic leukemia (JMML), B cell acute lymphoblastic leukemia, and acute myeloid leukemia (25, 26). In addition, activating mutations in SHP-2 have been identified in sporadic solid tumors (27). The SHP-2 mutations appear to play a causal role in the development of these diseases as SHP-2 mutations and other JMML-associated Ras or Neurofibromatosis 1 mutations are mutually exclusive in the patients (24–27). Moreover, single SHP-2 gain-of-function (GOF) mutations are sufficient to induce Noonan syndrome, cytokine hypersensitivity in hematopoietic progenitor cells, and JMML-like myeloproliferative disease in mice (28–32). Gain-of-function cell models derived from the newly available SHP-2 GOF mutation (D61G) knock-in mice (28) now provide us with a good opportunity to clarify the role of SHP-2 in cell motility. Unlike the dominant negative approach in which overexpression of mutant forms of SHP-2 generates complex effects, the SHP-2 D61G knock-in model eliminates this possibility as the mutant SHP-2 is expressed at the physiological level (28). Additionally, defining signaling functions of GOF mutant SHP-2 in cell movement can also help elucidate the molecular mechanisms by which SHP-2 mutations contribute to the relevant diseases.     

Hepatitis C Virus NS5A Protein Is a Substrate for the Peptidyl-prolyl cis/trans Isomerase Activity of Cyclophilins A and B

We report here a biochemical and structural characterization of domain 2 of the nonstructural 5A protein (NS5A) from the JFH1 Hepatitis C virus strain and its interactions with cyclophilins A and B (CypA and CypB). Gel filtration chromatography, circular dichroism spectroscopy, and finally NMR spectroscopy all indicate the natively unfolded nature of this NS5A-D2 domain. Because mutations in this domain have been linked to cyclosporin A resistance, we used NMR spectroscopy to investigate potential interactions between NS5A-D2 and cellular CypA and CypB. We observed a direct molecular interaction between NS5A-D2 and both cyclophilins. The interaction surface on the cyclophilins corresponds to their active site, whereas on NS5A-D2, it proved to be distributed over the many proline residues of the domain. NMR heteronuclear exchange spectroscopy yielded direct evidence that many proline residues in NS5A-D2 form a valid substrate for the enzymatic peptidyl-prolyl cis/trans isomerase (PPIase) activity of CypA and CypB.Hepatitis C virus (HCV)⁴ is a small, positive strand, RNA-enveloped virus belonging to the Flaviviridae family and the genus Hepacivirus. With 120–180 million chronically infected individuals worldwide, hepatitis C virus infection represents a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (1). The HCV viral genome (∼9.6 kb) codes for a unique polyprotein of ∼3000 amino acids (recently reviewed in Refs. 2–4). Following processing via viral and cellular proteases, this polyprotein gives rise to at least 10 viral proteins, divided into structural (core, E1, and E2 envelope glycoproteins) and nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B). Nonstructural proteins are involved in polyprotein processing and viral replication. The set composed of NS3, NS4A, NS4B, NS5A, and NS5B constitutes the minimal protein component required for viral replication (5).Cyclophilins are cellular proteins that have been identified first as CsA-binding proteins (6). As FK506-binding proteins (FKBP) and parvulins, cyclophilins are peptidyl-prolyl cis/trans isomerases (PPIase) that catalyze the cis/trans isomerization of the peptide linkage preceding a proline (6, 7). Several subtypes of cyclophilins are present in mammalian cells (8). They share a high sequence homology and a well conserved three-dimensional structure but display significant differences in their primary cellular localization and in abundance (9). CypA, the most abundant of the cyclophilins, is primarily cytoplasmic, whereas CypB is directed to the endoplasmic reticulum lumen or the secretory pathway. CypD, on the other hand, is the mitochondrial cyclophilin. Cyclophilins are involved in numerous physiological processes such as protein folding, immune response, and apoptosis and also in the replication cycle of viruses including vaccinia virus, vesicular stomatitis virus, severe acute respiratory syndrome (SARS)-coronavirus, and human immunodeficiency virus (HIV) (for review see Ref. 10). For HIV, CypA has been shown to interact with the capsid domain of the HIV Gag precursor polyprotein (11). CypA thereby competes with capsid domain/TRIM5 interaction, resulting in a loss of the antiviral protective effect of the cellular restriction factor TRIM5α (12, 13). Moreover, it has been shown that CypA catalyzes the cis/trans isomerization of Gly²²¹-Pro²²² in the capsid domain and that it has functional consequences for HIV replication efficiency (14–16). For HCV, Watashi et al. (17) have described a molecular and functional interaction between NS5B, the viral RNA-dependent RNA polymerase (RdRp), and cyclophilin B (CypB). CypB may be a key regulator in HCV replication by modulating the affinity of NS5B for RNA. This regulation is abolished in the presence of cyclosporin A (CsA), an inhibitor of cyclophilins (6). These results provided for the first time a molecular mechanism for the early-on observed anti-HCV activity of CsA (18–20). Although this initial report suggests that only CypB would be involved in the HCV replication process (17), a growing number of studies have recently pointed out a role for other cyclophilins (21–25).In vitro selection of CsA-resistant HCV mutants indicated the importance of two HCV nonstructural proteins, NS5B and NS5A (26), with a preponderant effect for mutations in the C-terminal half of NS5A. NS5A is a large phosphoprotein (49 kDa), indispensable for HCV replication and particle assembly (27–29), but for which the exact function(s) in the HCV replication cycle remain to be elucidated. This nonstructural protein is anchored to the cytoplasmic leaflet of the endoplasmic reticulum membrane via an N-terminal amphipathic α-helix (residues 1–27) (30, 31). Its cytoplasmic sequence can be divided into three domains: D1 (residues 27–213), D2 (residues 250–342), and D3 (residues 356–447), all connected by low complexity sequences (32). D1, a zinc-binding domain, adopts a dimeric claw-shaped structure, which is proposed to interact with RNA (33, 34). NS5A-D2 is essential for HCV replication, whereas NS5A-D3 is a key determinant for virus infectious particle assembly (27, 35). NS5A-D2 and -D3, for which sequence conservation among HCV genotypes is significantly lower than for D1, have been proposed to be natively unfolded domains (28, 32). Molecular and structural characterization of NS5A-D2 from HCV genotype 1a has confirmed the disordered nature of this domain (36, 37).As it is still not clear which cyclophilins are cofactors for HCV replication, and as mutations in HCV NS5A protein have been associated with CsA resistance, we decided to examine the interaction between both CypA and CypB and domain 2 of the HCV NS5A protein. We first characterized, at the molecular level, NS5A-D2 from the HCV JFH1 infectious strain (genotype 2a) and showed by NMR spectroscopy that this natively unfolded domain indeed interacts with both cyclophilin A and cyclophilin B. Our NMR chemical shift mapping experiments indicated that the interaction occurs at the level of the cyclophilin active site, whereas it lacks a precise localization on NS5A-D2. A peptide derived from the only well conserved amino acid motif in NS5A-D2 did interact with cyclophilin A but only with a 10-fold lower affinity than the full domain. We concluded from this that the many proline residues form multiple anchoring points, especially when they adopt the cis conformation. NMR exchange spectroscopy further demonstrated that NS5A-D2 is a substrate for the PPIase activities of both CypA and CypB. Both the NS5A/cyclophilin interaction and the PPIase activity of the cyclophilins on NS5A-D2 were abolished by CsA, underscoring the specificity of the interaction.     

Mechanism of Sustained Activation of Ribosomal S6 Kinase (RSK) and ERK by Kaposi Sarcoma-associated Herpesvirus ORF45: MULTIPROTEIN COMPLEXES RETAIN ACTIVE PHOSPHORYLATED ERK AND RSK AND PROTECT THEM FROM DEPHOSPHORYLATION*

As obligate intracellular parasites, viruses exploit diverse cellular signaling machineries, including the mitogen-activated protein-kinase pathway, during their infections. We have demonstrated previously that the open reading frame 45 (ORF45) of Kaposi sarcoma-associated herpesvirus interacts with p90 ribosomal S6 kinases (RSKs) and strongly stimulates their kinase activities (Kuang, E., Tang, Q., Maul, G. G., and Zhu, F. (2008) J. Virol. 82 ,1838 -1850). Here, we define the mechanism by which ORF45 activates RSKs. We demonstrated that binding of ORF45 to RSK increases the association of extracellular signal-regulated kinase (ERK) with RSK, such that ORF45, RSK, and ERK formed high molecular mass protein complexes. We further demonstrated that the complexes shielded active pERK and pRSK from dephosphorylation. As a result, the complex-associated RSK and ERK were activated and sustained at high levels. Finally, we provide evidence that this mechanism contributes to the sustained activation of ERK and RSK in Kaposi sarcoma-associated herpesvirus lytic replication.The extracellular signal-regulated kinase (ERK)² mitogen-activated protein kinase (MAPK) signaling pathway has been implicated in diverse cellular physiological processes including proliferation, survival, growth, differentiation, and motility (1-4) and is also exploited by a variety of viruses such as Kaposi sarcoma-associated herpesvirus (KSHV), human cytomegalovirus, human immunodeficiency virus, respiratory syncytial virus, hepatitis B virus, coxsackie, vaccinia, coronavirus, and influenza virus (5-17). The MAPK kinases relay the extracellular signaling through sequential phosphorylation to an array of cytoplasmic and nuclear substrates to elicit specific responses (1, 2, 18). Phosphorylation of MAPK is reversible. The kinetics of deactivation or duration of signaling dictates diverse biological outcomes (19, 20). For example, sustained but not transient activation of ERK signaling induces the differentiation of PC12 cells into sympathetic-like neurons and transformation of NIH3T3 cells (20-22). During viral infection, a unique biphasic ERK activation has been observed for some viruses (an early transient activation triggered by viral binding or entry and a late sustained activation correlated with viral gene expression), but the responsible viral factors and underlying mechanism for the sustained ERK activation remain largely unknown (5, 8, 13, 23).The p90 ribosomal S6 kinases (RSKs) are a family of serine/threonine kinases that lie at the terminus of the ERK pathway (1, 24-26). In mammals, four isoforms are known, RSK1 to RSK4. Each one has two catalytically functional kinase domains, the N-terminal kinase domain (NTKD) and C-terminal kinase domain (CTKD) as well as a linker region between the two. The NTKD is responsible for phosphorylation of exogenous substrates, and the CTKD and linker region regulate RSK activation (1, 24, 25). In quiescent cells ERK binds to the docking site in the C terminus of RSK (27-29). Upon mitogen stimulation, ERK is activated by its upstream MAPK/ERK kinase (MEK). The active ERK phosphorylates Thr-359/Ser-363 of RSK in the linker region (amino acid numbers refer to human RSK1) and Thr-573 in the CTKD activation loop. The activated CTKD then phosphorylates Ser-380 in the linker region, creating a docking site for 3-phosphoinositide-dependent protein kinase-1. The 3-phosphoinositide-dependent protein kinase-1 phosphorylates Ser-221 of RSK in the activation loop and activates the NTKD. The activated NTKD autophosphorylates the serine residue near the ERK docking site, causing a transient dissociation of active ERK from RSK (25, 26, 28). The stimulation of quiescent cells by a mitogen such as epidermal growth factor or a phorbol ester such as 12-O-tetradecanoylphorbol-13-acetate (TPA) usually results in a transient RSK activation that lasts less than 30 min. RSKs have been implicated in regulating cell survival, growth, and proliferation. Mutation or aberrant expression of RSK has been implicated in several human diseases including Coffin-Lowry syndrome and prostate and breast cancers (1, 24, 25, 30-32).KSHV is a human DNA tumor virus etiologically linked to Kaposi sarcoma, primary effusion lymphoma, and a subset of multicentric Castleman disease (33, 34). Infection and reactivation of KSHV activate multiple MAPK pathways (6, 12, 35). Noticeably, the ERK/RSK activation is sustained late during KSHV primary infection and reactivation from latency (5, 6, 12, 23), but the mechanism of the sustained ERK/RSK activation is unclear. Recently, we demonstrated that ORF45, an immediate early and also virion tegument protein of KSHV, interacts with RSK1 and RSK2 and strongly stimulates their kinase activities (23). We also demonstrated that the activation of RSK plays an essential role in KSHV lytic replication (23). In the present study we determined the mechanism of ORF45-induced sustained ERK/RSK activation. We found that ORF45 increases the association of RSK with ERK and protects them from dephosphorylation, causing sustained activation of both ERK and RSK.     

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.