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.     

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.     

Loss of PINK1 Function Promotes Mitophagy through Effects on Oxidative Stress and Mitochondrial Fission

Mitochondrial dysregulation is strongly implicated in Parkinson disease. Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is neuroprotective, less is known about neuronal responses to loss of PINK1 function. We found that stable knockdown of PINK1 induced mitochondrial fragmentation and autophagy in SH-SY5Y cells, which was reversed by the reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1. Moreover, stable or transient overexpression of wild-type PINK1 increased mitochondrial interconnectivity and suppressed toxin-induced autophagy/mitophagy. Mitochondrial oxidant production played an essential role in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines. Autophagy/mitophagy served a protective role in limiting cell death, and overexpressing Parkin further enhanced this protective mitophagic response. The dominant negative Drp1 mutant inhibited both fission and mitophagy in PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting oxidative stress, suggesting active involvement of autophagy in morphologic remodeling of mitochondria for clearance. To summarize, loss of PINK1 function elicits oxidative stress and mitochondrial turnover coordinated by the autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may cooperate through different mechanisms to maintain mitochondrial homeostasis.Parkinson disease is an age-related neurodegenerative disease that affects ∼1% of the population worldwide. The causes of sporadic cases are unknown, although mitochondrial or oxidative toxins such as 1-methyl-4-phenylpyridinium, 6-hydroxydopamine (6-OHDA),³ and rotenone reproduce features of the disease in animal and cell culture models (1). Abnormalities in mitochondrial respiration and increased oxidative stress are observed in cells and tissues from parkinsonian patients (2, 3), which also exhibit increased mitochondrial autophagy (4). Furthermore, mutations in parkinsonian genes affect oxidative stress response pathways and mitochondrial homeostasis (5). Thus, disruption of mitochondrial homeostasis represents a major factor implicated in the pathogenesis of sporadic and inherited parkinsonian disorders (PD).The PARK6 locus involved in autosomal recessive and early-onset PD encodes for PTEN-induced kinase 1 (PINK1) (6, 7). PINK1 is a cytosolic and mitochondrially localized 581-amino acid serine/threonine kinase that possesses an N-terminal mitochondrial targeting sequence (6, 8). The primary sequence also includes a putative transmembrane domain important for orientation of the PINK1 domain (8), a conserved kinase domain homologous to calcium calmodulin kinases, and a C-terminal domain that regulates autophosphorylation activity (9, 10). Overexpression of wild-type PINK1, but not its PD-associated mutants, protects against several toxic insults in neuronal cells (6, 11, 12). Mitochondrial targeting is necessary for some (13) but not all of the neuroprotective effects of PINK1 (14), implicating involvement of cytoplasmic targets that modulate mitochondrial pathobiology (8). PINK1 catalytic activity is necessary for its neuroprotective role, because a kinase-deficient K219M substitution in the ATP binding pocket of PINK1 abrogates its ability to protect neurons (14). Although PINK1 mutations do not seem to impair mitochondrial targeting, PD-associated mutations differentially destabilize the protein, resulting in loss of neuroprotective activities (13, 15).Recent studies indicate that PINK1 and Parkin interact genetically (3, 16-18) to prevent oxidative stress (19, 20) and regulate mitochondrial morphology (21). Primary cells derived from PINK1 mutant patients exhibit mitochondrial fragmentation with disorganized cristae, recapitulated by RNA interference studies in HeLa cells (3).Mitochondria are degraded by macroautophagy, a process involving sequestration of cytoplasmic cargo into membranous autophagic vacuoles (AVs) for delivery to lysosomes (22, 23). Interestingly, mitochondrial fission accompanies autophagic neurodegeneration elicited by the PD neurotoxin 6-OHDA (24, 25). Moreover, mitochondrial fragmentation and increased autophagy are observed in neurodegenerative diseases including Alzheimer and Parkinson diseases (4, 26-28). Although inclusion of mitochondria in autophagosomes was once believed to be a random process, as observed during starvation, studies involving hypoxia, mitochondrial damage, apoptotic stimuli, or limiting amounts of aerobic substrates in facultative anaerobes support the concept of selective mitochondrial autophagy (mitophagy) (29, 30). In particular, mitochondrially localized kinases may play an important role in models involving oxidative mitochondrial injury (25, 31, 32).Autophagy is involved in the clearance of protein aggregates (33-35) and normal regulation of axonal-synaptic morphology (36). Chronic disruption of lysosomal function results in accumulation of subtly impaired mitochondria with decreased calcium buffering capacity (37), implicating an important role for autophagy in mitochondrial homeostasis (37, 38). Recently, Parkin, which complements the effects of PINK1 deficiency on mitochondrial morphology (3), was found to promote autophagy of depolarized mitochondria (39). Conversely, Beclin 1-independent autophagy/mitophagy contributes to cell death elicited by the PD toxins 1-methyl-4-phenylpyridinium and 6-OHDA (25, 28, 31, 32), causing neurite retraction in cells expressing a PD-linked mutation in leucine-rich repeat kinase 2 (40). Whereas properly regulated autophagy plays a homeostatic and neuroprotective role, excessive or incomplete autophagy creates a condition of “autophagic stress” that can contribute to neurodegeneration (28).As mitochondrial fragmentation (3) and increased mitochondrial autophagy (4) have been described in human cells or tissues of PD patients, we investigated whether or not the engineered loss of PINK1 function could recapitulate these observations in human neuronal cells (SH-SY5Y). Stable knockdown of endogenous PINK1 gave rise to mitochondrial fragmentation and increased autophagy and mitophagy, whereas stable or transient overexpression of PINK1 had the opposite effect. Autophagy/mitophagy was dependent upon increased mitochondrial oxidant production and activation of fission. The data indicate that PINK1 is important for the maintenance of mitochondrial networks, suggesting that coordinated regulation of mitochondrial dynamics and autophagy limits cell death associated with loss of PINK1 function.     

Structure of a Viral Cap-independent Translation Element That Functions via High Affinity Binding to the eIF4E Subunit of eIF4F

RNAs of many positive strand RNA viruses lack a 5′ cap structure and instead rely on cap-independent translation elements (CITEs) to facilitate efficient translation initiation. The mechanisms by which these RNAs recruit ribosomes are poorly understood, and for many viruses the CITE is unknown. Here we identify the first CITE of an umbravirus in the 3′-untranslated region of pea enation mosaic virus RNA 2. Chemical and enzymatic probing of the ∼100-nucleotide PEMV RNA 2 CITE (PTE), and mutagenesis revealed that it forms a long, bulged helix that branches into two short stem-loops, with a possible pseudoknot interaction between a C-rich bulge at the branch point and a G-rich bulge in the main helix. The PTE inhibited translation in trans, and addition of eIF4F, but not eIFiso4F, restored translation. Filter binding assays revealed that the PTE binds eIF4F and its eIF4E subunit with high affinity. Tight binding required an intact cap-binding pocket in eIF4E. Among many PTE mutants, there was a strong correlation between PTE-eIF4E binding affinity and ability to stimulate cap-independent translation. We conclude that the PTE recruits eIF4F by binding eIF4E. The PTE represents a different class of translation enhancer element, as defined by its structure and ability to bind eIF4E in the absence of an m⁷G cap.Regulation of translation occurs primarily at the initiation step. This involves recognition of the 5′ m⁷G(5′)ppp(5′)N cap structure on the mRNA by initiation factors, which recruit the ribosome to the 5′-end of the mRNA (1–5). The 5′ cap structure and the poly(A) tail are necessary for efficient recruitment of initiation factors on eukaryotic mRNAs (3, 6–8). The cap is recognized by the eIF4E subunit of eukaryotic translation initiation factor complex eIF4F (or the eIFiso4E subunit of eIFiso4F in higher plants). The poly(A) tail is recognized by poly(A)-binding protein. In plants, eIF4F is a heterodimer consisting of eIF4E and eIF4G, the core scaffolding protein to which the other factors bind. eIF4A, an ATPase/RNA helicase, interacts with eIF4F but is not part of the eIF4F heterodimer (9, 10). For translation initiation, the purpose of eIF4E is to bring eIF4G to the capped mRNA. eIF4G then recruits the 43 S ternary ribosomal complex via interaction with eIF3.The RNAs of many positive sense RNA viruses contain a cap-independent translation element (CITE)³ that allows efficient translation in the absence of a 5′ cap structure (11–13). In animal viruses and some plant viruses, the CITE is an internal ribosome entry site (IRES) located upstream of the initiation codon. Most viral IRESes neither interact with nor require eIF4E, because they lack the m⁷GpppN structure, which, until this report, was thought to be necessary for mRNA to bind eIF4E with high affinity (3, 14). Translation initiation efficiency of mRNA is also influenced by the length of, and the degree of secondary structure in the 5′ leader (15–17).Many uncapped plant viral RNAs harbor a CITE in the 3′-UTR that confers highly efficient translation initiation at the 5′-end of the mRNA (18–22). These 3′ CITEs facilitate ribosome entry and apparently conventional scanning at the 5′-end of the mRNA (17, 23, 24). A variety of unrelated structures has been found to function as 3′ CITEs, suggesting that they recruit the ribosome by different interactions with initiation factors (13).The factors with which a plant CITE interacts to recruit the ribosome have been identified for only a potyvirus, a luteovirus, and a satellite RNA. The 143-nt 5′-UTR CITE of the potyvirus, tobacco etch virus is an IRES that functions by binding of its AU-rich pseudoknot structure with eIF4G (25). It binds eIF4G with up to 30-fold greater affinity than eIFiso4G and does not require eIF4E for IRES activity. In addition to RNA elements, the genome-linked viral protein (VPg) of potyviruses may participate in cap-independent translation initiation by interacting with the eIF4E and eIFiso4E subunits of eIF4F and eIFiso4F, respectively (26–31). In contrast, the 130-nt cap-independent translation enhancer domain (TED) in the 3′-UTR of satellite tobacco necrosis virus (STNV) RNA forms a long bulged stem-loop, which interacts strongly with both eIF4F and eIFiso4F and weakly with their eIF4E and eIFiso4E subunits (32), suggesting that the TED requires the full eIF4F or eIFiso4F for a biologically relevant interaction. Barley yellow dwarf luteovirus (BYDV) and several other viruses, have a different structure, called a BYDV-like CITE (BTE), in the 3′-UTR. The BTE is characterized by a 17-nt conserved sequence incorporated in a structure with a variable number of stem-loops radiating from a central junction (20, 33, 34). It requires and binds the eIF4G subunit of eIF4F and does not bind free eIF4E, eIFiso4E, or eIFiso4G, although eIF4E slightly enhances the BTE-eIF4G interaction (35). Other 3′ CITEs have been identified, but the host factors with which they interact are unknown.Here we describe unprecedented factor interactions of a CITE found in an umbravirus and a panicovirus. Umbraviruses show strong similarity to the Luteovirus and Dianthovirus genera in (i) the sequence of the replication genes encoded by ORFs 1 and 2, (ii) the predicted structure of the frameshift signals required for translation of the RNA-dependent RNA polymerase from ORF 2 (36, 37), (iii) the absence of a poly(A) tail, and (iv) the lack of a 5′ cap structure (37, 38). Umbraviruses are unique in that they encode no coat protein. For the umbravirus pea enation mosaic virus 2 (PEMV-2), the coat protein is provided by PEMV-1, an enamovirus (39). Uncapped PEMV-2 RNA (PEMV RNA 2), transcribed in vitro, is infectious in pea (Pisum sativa),⁴ indicating it must be translated cap-independently. The 3′-UTRs of some umbraviruses such as Tobacco bushy top virus and Groundnut rosette virus harbor sequences resembling BYDV-like CITEs (BTE).⁵ However, no BTE is apparent in the 3′-UTR of PEMV RNA 2. In this report we identify a different class of CITE in the 705-nt long 3′-UTR of PEMV RNA 2, determine its secondary structure, which may include an unusual pseudoknot, and we show that, unlike any other natural uncapped RNA, it has a high affinity for eIF4E, which is necessary to facilitate cap-independent translation.     

The Cholinesterase-like Domain, Essential in Thyroglobulin Trafficking for Thyroid Hormone Synthesis, Is Required for Protein Dimerization

The carboxyl-terminal cholinesterase-like (ChEL) domain of thyroglobulin (Tg) has been identified as critically important in Tg export from the endoplasmic reticulum. In a number of human kindreds suffering from congenital hypothyroidism, and in the cog congenital goiter mouse and rdw rat dwarf models, thyroid hormone synthesis is inhibited because of mutations in the ChEL domain that block protein export from the endoplasmic reticulum. We hypothesize that Tg forms homodimers through noncovalent interactions involving two predicted α-helices in each ChEL domain that are homologous to the dimerization helices of acetylcholinesterase. This has been explored through selective epitope tagging of dimerization partners and by inserting an extra, unpaired Cys residue to create an opportunity for intermolecular disulfide pairing. We show that the ChEL domain is necessary and sufficient for Tg dimerization; specifically, the isolated ChEL domain can dimerize with full-length Tg or with itself. Insertion of an N-linked glycan into the putative upstream dimerization helix inhibits homodimerization of the isolated ChEL domain. However, interestingly, co-expression of upstream Tg domains, either in cis or in trans, overrides the dimerization defect of such a mutant. Thus, although the ChEL domain provides a nidus for Tg dimerization, interactions of upstream Tg regions with the ChEL domain actively stabilizes the Tg dimer complex for intracellular transport.The synthesis of thyroid hormone in the thyroid gland requires secretion of thyroglobulin (Tg)² to the apical luminal cavity of thyroid follicles (1). Once secreted, Tg is iodinated via the activity of thyroid peroxidase (2). A coupling reaction involving a quinol-ether linkage especially engages di-iodinated tyrosyl residues 5 and 130 to form thyroxine within the amino-terminal portion of the Tg polypeptide (3, 4). Preferential iodination of Tg hormonogenic sites is dependent not on the specificity of the peroxidase (5) but upon the native structure of Tg (6, 7). To date, no other thyroidal proteins have been shown to effectively substitute in this role for Tg.The first 80% of the primary structure of Tg (full-length murine Tg: 2,746 amino acids) involves three regions called I-II-III comprised of disulfide-rich repeat domains held together by intradomain disulfide bonds (8, 9). The final 581 amino acids of Tg are strongly homologous to acetylcholinesterase (10–12). Rate-limiting steps in the overall process of Tg secretion involve its structural maturation within the endoplasmic reticulum (ER) (13). Interactions between regions I-II-III and the cholinesterase-like (ChEL) domain have recently been suggested to be important in this process, with ChEL functioning as an intramolecular chaperone and escort for I-II-III (14). In addition, Tg conformational maturation culminates in Tg homodimerization (15, 16) with progression to a cylindrical, and ultimately, a compact ovoid structure (17–19).In human congenital hypothyroidism with deficient Tg, the ChEL domain is a commonly affected site of mutation, including the recently described A2215D (20, 21), R2223H (22), G2300D, R2317Q (23), G2355V, G2356R, and the skipping of exon 45 (which normally encodes 36 amino acids), as well as the Q2638stop mutant (24) (in addition to polymorphisms including P2213L, W2482R, and R2511Q that may be associated with thyroid overgrowth (25)). As best as is currently known, all of the congenital hypothyroidism-inducing Tg mutants are defective for intracellular transport (26). A homozygous G2300R mutation (equivalent to residue 2,298 of mouse Tg) in the ChEL domain is responsible for congenital hypothyroidism in rdw rats (27, 28), whereas we identified the Tg-L2263P point mutation as the cause of hypothyroidism in the cog mouse (29). Such mutations perturb intradomain structure (30), and interestingly, block homodimerization (31). Acquisition of quaternary structure has long been thought to be required for efficient export from the ER (32) as exemplified by authentic acetylcholinesterase (33, 34) in which dimerization enhances protein stability and export (35).Tg comprised only of regions I-II-III (truncated to lack the ChEL domain) is blocked within the ER (30), whereas a secretory version of the isolated ChEL domain of Tg devoid of I-II-III undergoes rapid and efficient intracellular transport and secretion (14). A striking homology positions two predicted α-helices of the ChEL domain to the identical relative positions of the dimerization helices in acetylcholinesterase. This raises the possibility that ChEL may serve as a homodimerization domain for Tg, providing a critical function in maturation for Tg transport to the site of thyroid hormone synthesis (1).In this study, we provide unequivocal evidence for homodimerization of the ChEL domain and “hetero”-dimerization of that domain with full-length Tg, and we provide significant evidence that the predicted ChEL dimerization helices provide a nidus for Tg assembly. On the other hand, our data also suggest that upstream Tg regions known to interact with ChEL (14) actively stabilize the Tg dimer complex. Together, I-II-III and ChEL provide unique contributions to the process of intracellular transport of Tg through the secretory pathway.     

Intersectin Regulates Dendritic Spine Development and Somatodendritic Endocytosis but Not Synaptic Vesicle Recycling in Hippocampal Neurons

Intersectin-short (intersectin-s) is a multimodule scaffolding protein functioning in constitutive and regulated forms of endocytosis in non-neuronal cells and in synaptic vesicle (SV) recycling at the neuromuscular junction of Drosophila and Caenorhabditis elegans. In vertebrates, alternative splicing generates a second isoform, intersectin-long (intersectin-l), that contains additional modular domains providing a guanine nucleotide exchange factor activity for Cdc42. In mammals, intersectin-s is expressed in multiple tissues and cells, including glia, but excluded from neurons, whereas intersectin-l is a neuron-specific isoform. Thus, intersectin-I may regulate multiple forms of endocytosis in mammalian neurons, including SV endocytosis. We now report, however, that intersectin-l is localized to somatodendritic regions of cultured hippocampal neurons, with some juxtanuclear accumulation, but is excluded from synaptophysin-labeled axon terminals. Consistently, intersectin-l knockdown (KD) does not affect SV recycling. Instead intersectin-l co-localizes with clathrin heavy chain and adaptor protein 2 in the somatodendritic region of neurons, and its KD reduces the rate of transferrin endocytosis. The protein also co-localizes with F-actin at dendritic spines, and intersectin-l KD disrupts spine maturation during development. Our data indicate that intersectin-l is indeed an important regulator of constitutive endocytosis and neuronal development but that it is not a prominent player in the regulated endocytosis of SVs.Clathrin-mediated endocytosis (CME)⁴ is a major mechanism by which cells take up nutrients, control the surface levels of multiple proteins, including ion channels and transporters, and regulate the coupling of signaling receptors to downstream signaling cascades (1-5). In neurons, CME takes on additional specialized roles; it is an important process regulating synaptic vesicle (SV) availability through endocytosis and recycling of SV membranes (6, 7), it shapes synaptic plasticity (8-10), and it is crucial in maintaining synaptic membranes and membrane structure (11).Numerous endocytic accessory proteins participate in CME, interacting with each other and with core components of the endocytic machinery such as clathrin heavy chain (CHC) and adaptor protein-2 (AP-2) through specific modules and peptide motifs (12). One such module is the Eps15 homology domain that binds to proteins bearing NPF motifs (13, 14). Another is the Src homology 3 (SH3) domain, which binds to proline-rich domains in protein partners (15). Intersectin is a multimodule scaffolding protein that interacts with a wide range of proteins, including several involved in CME (16). Intersectin has two N-terminal Eps15 homology domains that are responsible for binding to epsin, SCAMP1, and numb (17-19), a central coil-coiled domain that interacts with Eps15 and SNAP-23 and -25 (17, 20, 21), and five SH3 domains in its C-terminal region that interact with multiple proline-rich domain proteins, including synaptojanin, dynamin, N-WASP, CdGAP, and mSOS (16, 22-25). The rich binding capability of intersectin has linked it to various functions from CME (17, 26, 27) and signaling (22, 28, 29) to mitogenesis (30, 31) and regulation of the actin cytoskeleton (23).Intersectin functions in SV recycling at the neuromuscular junction of Drosophila and C. elegans where it acts as a scaffold, regulating the synaptic levels of endocytic accessory proteins (21, 32-34). In vertebrates, the intersectin gene is subject to alternative splicing, and a longer isoform (intersectin-l) is generated that is expressed exclusively in neurons (26, 28, 35, 36). This isoform has all the binding modules of its short (intersectin-s) counterpart but also has additional domains: a DH and a PH domain that provide guanine nucleotide exchange factor (GEF) activity specific for Cdc42 (23, 37) and a C2 domain at the C terminus. Through its GEF activity and binding to actin regulatory proteins, including N-WASP, intersectin-l has been implicated in actin regulation and the development of dendritic spines (19, 23, 24). In addition, because the rest of the binding modules are shared between intersectin-s and -l, it is generally thought that the two intersectin isoforms have the same endocytic functions. In particular, given the well defined role for the invertebrate orthologs of intersectin-s in SV endocytosis, it is thought that intersectin-l performs this role in mammalian neurons, which lack intersectin-s. Defining the complement of intersectin functional activities in mammalian neurons is particularly relevant given that the protein is involved in the pathophysiology of Down syndrome (DS). Specifically, the intersectin gene is localized on chromosome 21q22.2 and is overexpressed in DS brains (38). Interestingly, alterations in endosomal pathways are a hallmark of DS neurons and neurons from the partial trisomy 16 mouse, Ts65Dn, a model for DS (39, 40). Thus, an endocytic trafficking defect may contribute to the DS disease process.Here, the functional roles of intersectin-l were studied in cultured hippocampal neurons. We find that intersectin-l is localized to the somatodendritic regions of neurons, where it co-localizes with CHC and AP-2 and regulates the uptake of transferrin. Intersectin-l also co-localizes with actin at dendritic spines and disrupting intersectin-l function alters dendritic spine development. In contrast, intersectin-l is absent from presynaptic terminals and has little or no role in SV recycling.     

Regulated Proteolysis of Nonmuscle Myosin IIA Stimulates Osteoclast Fusion

The nonmuscle myosin IIA heavy chain (Myh9) is strongly associated with adhesion structures of osteoclasts. In this study, we demonstrate that during osteoclastogenesis, myosin IIA heavy chain levels are temporarily suppressed, an event that stimulates the onset of cell fusion. This suppression is not mediated by changes in mRNA or translational levels but instead is due to a temporary increase in the rate of myosin IIA degradation. Intracellular activity of cathepsin B is significantly enhanced at the onset of osteoclast precursor fusion, and specific inhibition of its activity prevents myosin IIA degradation. Further, treatment of normal cells with cathepsin B inhibitors during the differentiation process reduces cell fusion and bone resorption capacity, whereas overexpression of cathepsin B enhances fusion. Ongoing suppression of the myosin IIA heavy chain via RNA interference results in formation of large osteoclasts with significantly increased numbers of nuclei, whereas overexpression of myosin IIA results in less osteoclast fusion. Increased multinucleation caused by myosin IIA suppression does not require RANKL. Further, knockdown of myosin IIA enhances cell spreading and lessens motility. These data taken together strongly suggest that base-line expression of nonmuscle myosin IIA inhibits osteoclast precursor fusion and that a temporary, cathepsin B-mediated decrease in myosin IIA levels triggers precursor fusion during osteoclastogenesis.The final stages of osteoclastogenesis involve fusion of differentiated precursors from the monocyte/macrophage lineage (1). Although the membrane structural components regulating preosteoclast fusion are not well understood, in recent years a number of candidate cell surface molecules have been implicated, including receptors CD44 (2, 3), CD47 and its ligand macrophage fusion receptor (also known as signal regulatory protein α) (4–6), the purinergic receptor P2X₇ (7), and the disintegrin and metalloproteinase ADAM8 (8). A recently identified receptor, the dendritic cell-specific transmembrane protein, is essential for osteoclast fusion both in vitro and in vivo (9, 10). More recently, the d2 subunit of proton-translocating vacuolar proton-translocating ATPases, a membrane subunit isoform expressed predominantly in osteoclasts, similarly was demonstrated to be required for fusion in vitro and in vivo (11). However, elucidation of the mechanisms by which these molecules may mediate cell fusion has proved to be difficult.The mammalian class II myosin family consists of distinct isoforms expressed in skeletal, smooth, and cardiac muscle, as well as three nonmuscle forms designated IIA, IIB, and IIC (12–14). Although all class II molecules are composed of two heavy chains, two essential light chains, and two regulatory chains, their unique activities are a function of their particular heavy chain isoforms. Although the nonmuscle heavy chain isoforms share extensive structural homology, they have been shown to demonstrate distinct patterns of expression (15–18), enzyme kinetics and activation (12, 19–21), and cellular function (22–24). Knock-out of either myosin IIA or IIB results in embryonic lethality, although death derives from defects unique to each isoform (25, 26). In vitro, myosin IIA, a target of Rho kinase, has been shown to be involved in a wide variety of cellular functions, including cytokinesis, cell contractility, and adhesion and motility.The actin cytoskeleton of osteoclasts possesses features unlike those of most mammalian cell types. First, osteoclasts do not possess stress fibers but instead form a meshwork of fine actin filaments throughout the cell (27–29). Osteoclasts express unusual attachment structures typified by the podosome, a form of adhesion structure most typically present in cells of the monocyte/macrophage lineage, dendritic cells, and smooth muscle cells. Podosomes are integrin-based cell-matrix contact structures that are notable for the presence of a short (0.5–1.0 μm) F-actin core surrounded by a ring of adaptor proteins, kinases, small GTPases, and regulators of endocytosis (30, 31). When cultured on glass, mature osteoclasts generate a belt of podosomes at the cell periphery. However, when cultured on bone, osteoclasts form a dense ring of podosome-like structures that is usually internal to the cell margins (32). This region, termed the sealing zone, surrounds a specialized membrane domain termed the ruffled border, from which protons and proteases are secreted to induce resorption of bone (1). We previously demonstrated that myosins IIA and IIB localize to distinct subcellular regions within osteoclasts, with MyoIIA² strongly segregating to both podosomes and the actin ring of the sealing zone (28). Because of this distribution into osteoclast adhesion structures and findings in other cells showing MyoIIA to be associated with dynamic Rho-kinase-dependent functions, such as adhesion and locomotion, we hypothesized that MyoIIA may play a vital role in cell motility and the bone resorption function. In this study, we examined cellular expression of MyoIIA during osteoclastogenesis and, along with RNA interference-mediated suppression of the protein, have confirmed its role in cell spreading, motility, and sealing zone formation. However, this study also unexpectedly revealed a role for MyoIIA in regulating preosteoclast fusion during osteoclastogenesis.     

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.     

Mapping Daunorubicin-binding Sites in the ATP-binding Cassette Transporter MsbA Using Site-specific Quenching by Spin Labels

ATP-binding cassette (ABC) transporters transduce the free energy of ATP hydrolysis to power the mechanical work of substrate translocation across cell membranes. MsbA is an ABC transporter implicated in trafficking lipid A across the inner membrane of Escherichia coli. It has sequence similarity and overlapping substrate specificity with multidrug ABC transporters that export cytotoxic molecules in humans and prokaryotes. Despite rapid advances in structure determination of ABC efflux transporters, little is known regarding the location of substrate-binding sites in the transmembrane segment and the translocation pathway across the membrane. In this study, we have mapped residues proximal to the daunorubicin (DNR)-binding site in MsbA using site-specific, ATP-dependent quenching of DNR intrinsic fluorescence by spin labels. In the nucleotide-free MsbA intermediate, DNR-binding residues cluster at the cytoplasmic end of helices 3 and 6 at a site accessible from the membrane/water interface and extending into an aqueous chamber formed at the interface between the two transmembrane domains. Binding of a nonhydrolyzable ATP analog inverts the transporter to an outward-facing conformation and relieves DNR quenching by spin labels suggesting DNR exclusion from proximity to the spin labels. The simplest model consistent with our data has DNR entering near an elbow helix parallel to the water/membrane interface, partitioning into the open chamber, and then translocating toward the periplasm upon ATP binding.ATP-binding cassette (ABC)² transporters transduce the energy of ATP hydrolysis to power the movement of a wide range of substrates across the cell membranes (1, 2). They constitute the largest family of prokaryotic transporters, import essential cell nutrients, flip lipids, and export toxic molecules (3). Forty eight human ABC transporters have been identified, including ABCB1, or P-glycoprotein, which is implicated in cross-resistance to drugs and cytotoxic molecules (4, 5). Inherited mutations in these proteins are linked to diseases such as cystic fibrosis, persistent hypoglycemia of infancy, and immune deficiency (6).The functional unit of an ABC transporter consists of four modules. Two highly conserved ABCs or nucleotide-binding domains (NBDs) bind and hydrolyze ATP to supply the active energy for transport (7). ABCs drive the mechanical work of proteins with diverse functions ranging from membrane transport to DNA repair (3, 5). Substrate specificity is determined by two transmembrane domains (TMDs) that also provide the translocation pathway across the bilayer (7). Bacterial ABC exporters are expressed as monomers, each consisting of one NBD and one TMD, that dimerize to form the active transporter (3). The number of transmembrane helices and their organization differ significantly between ABC importers and exporters reflecting the divergent structural and chemical nature of their substrates (1, 8, 9). Furthermore, ABC exporters bind substrates directly from the cytoplasm or bilayer inner leaflet and release them to the periplasm or bilayer outer leaflet (10, 11). In contrast, bacterial importers have their substrates delivered to the TMD by a dedicated high affinity substrate-binding protein (12).In Gram-negative bacteria, lipid A trafficking from its synthesis site on the inner membrane to its final destination in the outer membrane requires the ABC transporter MsbA (13). Although MsbA has not been directly shown to transport lipid A, suppression of MsbA activity leads to cytoplasmic accumulation of lipid A and inhibits bacterial growth strongly suggesting a role in translocation (14-16). In addition to this role in lipid A transport, MsbA shares sequence similarity with multidrug ABC transporters such as human ABCB1, LmrA of Lactococcus lactis, and Sav1866 of Staphylococcus aureus (16-19). ABCB1, a prototype of the ABC family, is a plasma membrane protein whose overexpression provides resistance to chemotherapeutic agents in cancer cells (1). LmrA and MsbA have overlapping substrate specificity with ABCB1 suggesting that both proteins can function as drug exporters (18, 20). Indeed, cells expressing MsbA confer resistance to erythromycin and ethidium bromide (21). MsbA can be photolabeled with the ABCB1/LmrA substrate azidopine and can transport Hoechst 33342 ({"type":"entrez-nucleotide","attrs":{"text":"H33342","term_id":"978759","term_text":"H33342"}}H33342) across membrane vesicles in an energy-dependent manner (21).The structural mechanics of ABC exporters was revealed from comparison of the MsbA crystal structures in the apo- and nucleotide-bound states as well as from analysis by spin labeling EPR spectroscopy in liposomes (17, 19, 22, 23). The energy harnessed from ATP binding and hydrolysis drives a cycle of NBD association and dissociation that is transmitted to induce reorientation of the TMD from an inward- to outward-facing conformation (17, 19, 22). Large amplitude motion closes the cytoplasmic end of a chamber found at the interface between the two TMDs and opens it to the periplasm (23). These rearrangements lead to significant changes in chamber hydration, which may drive substrate translocation (22).Substrate binding must precede energy input, otherwise the cycle is futile, wasting the energy of ATP hydrolysis without substrate extrusion (7). Consistent with this model, ATP binding reduces ABCB1 substrate affinity, potentially through binding site occlusion (24-26). Furthermore, the TMD substrate-binding event signals the NBD to stimulate ATP hydrolysis increasing transport efficiency (1, 27, 28). However, there is a paucity of information regarding the location of substrate binding, the transport pathway, and the structural basis of substrate recognition by ABC exporters. In vitro studies of MsbA substrate specificity identify a broad range of substrates that stimulate ATPase activity (29). In addition to the putative physiological substrates lipid A and lipopolysaccharide (LPS), the ABCB1 substrates Ilmofosine, {"type":"entrez-nucleotide","attrs":{"text":"H33342","term_id":"978759","term_text":"H33342"}}H33342, and verapamil differentially enhance ATP hydrolysis of MsbA (29, 30). Intrinsic MsbA tryptophan (Trp) fluorescence quenching by these putative substrate molecules provides further support of interaction (29).Extensive biochemical analysis of ABCB1 and LmrA provides a general model of substrate binding to ABC efflux exporters. This so-called “hydrophobic cleaner model” describes substrates binding from the inner leaflet of the bilayer and then translocating through the TMD (10, 31, 32). These studies also identified a large number of residues involved in substrate binding and selectivity (33). When these crucial residues are mapped onto the crystal structures of MsbA, a subset of homologous residues clusters to helices 3 and 6 lining the putative substrate pathway (34). Consistent with a role in substrate binding and specificity, simultaneous replacement of two serines (Ser-289 and Ser-290) in helix 6 of MsbA reduces binding and transport of ethidium and taxol, although {"type":"entrez-nucleotide","attrs":{"text":"H33342","term_id":"978759","term_text":"H33342"}}H33342 and erythromycin interactions remain unaffected (34).The tendency of lipophilic substrates to partition into membranes confounds direct analysis of substrate interactions with ABC exporters (35, 36). Such partitioning may promote dynamic collisions with exposed Trp residues and nonspecific cross-linking in photo-affinity labeling experiments. In this study, we utilize a site-specific quenching approach to identify residues in the vicinity of the daunorubicin (DNR)-binding site (37). Although the data on DNR stimulation of ATP hydrolysis is inconclusive (20, 29, 30), the quenching of MsbA Trp fluorescence suggests a specific interaction. Spin labels were introduced along transmembrane helices 3, 4, and 6 of MsbA to assess their ATP-dependent quenching of DNR fluorescence. Residues that quench DNR cluster along the cytoplasmic end of helices 3 and 6 consistent with specific binding of DNR. Furthermore, many of these residues are not lipid-exposed but face the putative substrate chamber formed between the two TMDs. These residues are proximal to two Trps, which likely explains the previously reported quenching (29). Our results suggest DNR partitions to the membrane and then binds MsbA in a manner consistent with the hydrophobic cleaner model. Interpretation in the context of the crystal structures of MsbA identifies a putative translocation pathway through the transmembrane segment.     

A Role for the Proton-coupled Folate Transporter (PCFT-SLC46A1) in Folate Receptor-mediated Endocytosis

Recently, this laboratory identified a proton-coupled folate transporter (PCFT), with optimal activity at low pH. PCFT is critical to intestinal folate absorption and transport into the central nervous system because there are loss-of-function mutations in this gene in the autosomal recessive disorder, hereditary folate malabsorption. The current study addresses the role PCFT might play in another transport pathway, folate receptor (FR)-mediated endocytosis. FRα cDNA was transfected into novel PCFT⁺ and PCFT^– HeLa sublines. FRα was shown to bind and trap folates in vesicles but with minimal export into the cytosol in PCFT^– cells. Cotransfection of FRα and PCFT resulted in enhanced folate transport into cytosol as compared with transfection of FRα alone. Probenecid did not inhibit folate binding to FR, but inhibited PCFT-mediated transport at endosomal pH, and blocked FRα-mediated transport into the cytosol. FRα and PCFT co-localized to the endosomal compartment. These observations (i) indicate that PCFT plays a role in FRα-mediated endocytosis by serving as a route of export of folates from acidified endosomes and (ii) provide a functional role for PCFT in tissues in which it is expressed, such as the choroid plexus, where the extracellular milieu is at neutral pH.Loss of function mutations of the proton-coupled folate transporter (PCFT),² which functions optimally at low pH, are the molecular basis for the autosomal recessive disorder, hereditary folate malabsorption (HFM) (1–4). Infants present with this disorder several months after birth with marked folate deficiency anemia, hypogammaglobulinemia with immune deficiency and infections, neurological deficits, and often seizures (5). PCFT is highly expressed at the apical brush-border membrane of the duodenum and proximal jejunum (6–9) where the pH at the microclimate of the surface of this epithelium is low (pH 5.8–6.0), and folates are absorbed (1, 7, 10, 11). Hence, the failure to absorb folates in the absence of this transporter in HFM is expected. However, PCFT expression, and its associated folate transport activity at low pH, is observed in many tissues where the transport interface is presumed to be at pH 7.4 (12). Of particular interest is the mechanism by which PCFT mediates transport of folates into the central nervous system (CNS) where this transporter is expressed in brain and choroid plexus (1, 7, 13). Transport into the CNS is impaired in patients with HFM who have very low cerebrospinal fluid (CSF) folate levels and marked reversal of the blood:CSF folate gradient which is normally 2–3:1 (5).Folates are also transported into cells by a receptor-mediated process. Folate receptor-α (FRα) is anchored to cell membranes via a glycosylphosphatidylinositol domain. Uptake begins with folate binding to receptor at the cell surface followed by invagination of the membrane and the formation of endosomes that traffic along microtubules to a perinuclear compartment before returning to the plasma membrane (14–16). During transit in the cytoplasm, endosomes acidify to a pH of ∼6.0–6.5 (17), folate is released from the receptor and exported from the intact endosome into the cytoplasm. This putative exporter was shown to require a trans-endosomal pH gradient (18–20).The current report addresses the hypothesis that PCFT is an endosomal folate exporter and thereby plays a role in FRα-mediated endocytosis (1, 2, 21, 22), that the ubiquitous expression of PCFT in mammalian tissues may be related to this function, and that loss of this function may be a basis for the low CSF folate levels in HFM. The experimental approach utilized a series of HeLa sublines, developed in this laboratory, in which constitutive expression of FRα is negligible. HeLa R5 cells lack reduced folate carrier (RFC) function due to a genomic deletion of this gene (23). A derivative of R5 cells, HeLa R1-11 cells lack, in addition, PCFT expression, while an R1-11 revertant re-expresses PCFT (24). The impact of PCFT on FRα-mediated endocytosis, achieved by transfection of the receptor into these cell lines, was assessed under conditions in which there was negligible PCFT-mediated transport directly across the plasma membrane into cells.