c-Src Associates with ErbB2 through an Interaction between Catalytic Domains and Confers Enhanced Transforming Potential

Previous studies have demonstrated that c-Src tyrosine kinase interacts specifically with ErbB2, but not with other members of the epidermal growth factor receptor (EGFR) family. To identify the site of interaction, we recently used a chimeric EGFR/ErbB2 receptor approach to show that c-Src requires the kinase region of ErbB2 for binding. Here, we demonstrate that retention of a conserved amino acid motif surrounding tyrosine 877 (referred to here as EGFR^YHAD) is sufficient to confer binding to c-Src. Surprisingly the association of c-Src was not dependent on its SH2 or SH3 domain or on the phosphorylation or kinase activity of the receptor. We further show that the chimeric EGFRs that contain the Y877 motif are transforming in vitro and in vivo following ligand stimulation. Transformation was also partially dependent on sustained activation of Stat3. Finally, we demonstrate that EGFRs with mutations in the catalytic domain, originally identified in lung cancer and conferring increased sensitivity to gefitinib and erlotinib, two EGFR kinase inhibitors, gained the capacity to bind c-Src. Moreover, transformation by these EGFR mutants was inhibited by Src inhibitors regardless of their sensitivities to gefitinib and erlotinib. These observations have important implications for understanding the molecular basis for resistance to EGFR inhibitors and implicate c-Src as a critical signaling molecule in EGFR mutant-induced transformation.The epidermal growth factor receptor (EGFR) family is comprised of four members, EGFR, ErbB2, ErbB3, and ErbB4, with distinct ligand specificities, which, upon homo- or heterodimerization after ligand binding, autophosphorylate and recruit different effector proteins to specific tyrosine residues located in their cytoplasmic tails. These signaling molecules, which are either adapter molecules that recruit other kinases or kinases themselves, mediate diverse functions, such as proliferation, growth, and survival (27). There are now several pieces of evidence demonstrating that these growth factor receptors are mutated or overexpressed in a variety of different cancers, including salivary gland adenocarcinoma (44), breast cancer (47), esophageal squamous carcinoma (22), bladder cancer (58), and lung cancer (57). Accordingly, ErbB2 is overexpressed in 20 to 30% of all human breast cancer, which correlates with poor prognosis, and in 40 to 60% of ductal carcinoma in situ (19). ErbB2 is 100-fold more potent in its transforming ability than ErbB1/EGFR, although the two receptors are 85% homologous (14, 15). Breast carcinoma cells devoid of ErbB2, but not other ErbB receptor family members, are defective in cell invasion upon EGF ligand stimulation (49). In fact, ErbB2 could induce cell migration when overexpressed in cells devoid of any other ErbB receptors. In a three-dimensional cell culture system, overexpression of ErbB2, but not EGFR, disrupts mammary acinus structure by reinitiating cell proliferation, leading to an absence of lumen and disruption of tight junctions and of cell polarity, although the cells still lack invasive properties (31).Src is a nonreceptor tyrosine kinase implicated in signal transduction pathways downstream of multiple receptors, such as platelet-derived growth factor, insulin receptor, G-coupled receptors, and ErbB family receptors, where it regulates a wide variety of cellular functions that include proliferation, migration, and apoptosis (17). Src tyrosine kinase activity is sporadically increased in many cases of human cancer, including colon and breast cancer (10, 38, 52). Moreover, Src kinase activity is elevated in ErbB2-induced mammary tumors (33). Direct evidence supporting a role in mammary tumor progression derives from observations made in transgenic mice. Constitutive activation of c-Src in mammary epithelia led to frequent mammary epithelial hyperplasias, which occasionally developed into solid tumors (54). Conversely, deletion of c-Src in a mouse mammary tumor virus/polyomavirus middle T-antigen (PyMT) transgenic strain abrogates mammary tumor formation (21).c-Src is also an important player downstream of the EGFR family. Phosphorylation of several tyrosine residues within the EGFR has been demonstrated to be increased following c-Src overexpression both in vitro and in vivo, suggesting that c-Src is required for full biological response following EGF stimulation (29, 51). In addition to EGFR, c-Src specifically interacts with tyrosine-phosphorylated ErbB2 in ErbB2-induced mammary tumors. This association was further demonstrated to result in enhanced c-Src kinase activity (3, 28, 34, 35). More recently, using chimeric EGF/ErbB2 receptors, we demonstrated that c-Src specifically associates with ErbB2, but not with other ErbB family members. c-Src was demonstrated to specifically associate with the ErbB2 kinase domain (24). Moreover, the chimeric EGFR that contained the c-Src binding site was able to disrupt cell polarity and cell-cell junctions to induce epithelial cell scattering in a three-dimensional cell culture system in a MAPK-dependent manner (24).Here, we demonstrate that c-Src association with ErbB2 is conformation dependent and that the residues necessary for interaction are centered around Y877 in the kinase domain of ErbB2, an association that is further strengthened by residues located in the amino-terminal part of the kinase domain. This association was not dependent on the SH2 or SH3 domain or the kinase activity of c-Src or ErbB2. We further show that mammary epithelial cells expressing the EGFR/ErbB2 chimeric receptors that have regained the capacity to associate with c-Src have disrupted epithelial polarity that is correlated with enhanced transforming potential, an effect dependent on c-Src kinase activity and Stat3 activation. Finally, we show that mutant EGFRs isolated from lung adenocarcinomas have the capacity to associate with c-Src and that these EGFR mutants require Src kinase activity for transformation.     

Epidermal Growth Factor Receptor Activation Remodels the Plasma Membrane Lipid Environment To Induce Nanocluster Formation

Signal transduction is regulated by the lateral segregation of proteins into nanodomains on the plasma membrane. However, the molecular mechanisms that regulate the lateral segregation of cell surface receptors, such as receptor tyrosine kinases, upon ligand binding are unresolved. Here we used high-resolution spatial mapping to investigate the plasma membrane nanoscale organization of the epidermal growth factor (EGF) receptor (EGFR). Our data demonstrate that in serum-starved cells, the EGFR exists in preformed, cholesterol-dependent, actin-independent nanoclusters. Following stimulation with EGF, the number and size of EGFR nanoclusters increase in a time-dependent manner. Our data show that the formation of EGFR nanoclusters requires receptor tyrosine kinase activity. Critically, we show for the first time that production of phosphatidic acid by phospholipase D2 (PLD2) is essential for ligand-induced EGFR nanocluster formation. In accordance with its crucial role in regulating EGFR nanocluster formation, we demonstrate that modulating PLD2 activity tunes the degree of EGFR nanocluster formation and mitogen-activated protein kinase signal output. Together, these data show that EGFR activation drives the formation of signaling domains by regulating the production of critical second-messenger lipids and modifying the local membrane lipid environment.The epidermal growth factor (EGF) receptor (EGFR) is a single transmembrane domain protein that possesses intrinsic tyrosine kinase (TK) activity. Ligand binding to the extracellular domain induces conformational changes that promote activation of the intracellular TK domain. The kinase domain then autophosphorylates a number of tyrosine residues in the C-terminal region of the protein, creating docking sites for adapter and effector proteins. Thus, the active form of the EGFR could reasonably be expected to be a dimer. However, recent studies using single-molecule imaging, image correlation spectroscopy (ICS), fluorescence correlation spectroscopy (FCS), and immunoelectron microscopy (immuno-EM) show that the EGFR is, in fact, nonrandomly organized into oligomers on the plasma membrane (6, 7, 16, 34, 44). ICS measurements estimate that, in the absence of ligand, there are, on average, 2.2 EGFRs per cluster, which increases to 3.7 receptors per cluster upon stimulation (7). Single-molecule tracking experiments also suggest that unliganded EGFRs continually fluctuate between monomers and dimers that are primed for activation (5). Furthermore, the organization of the EGFR is dynamic and clustering of the EGFR increases over time after EGF stimulation (7, 16). However, neither the precise role of EGFR oligomerization in signal transduction nor the mechanisms driving oligomer formation have been resolved.The organization of the EGFR into oligomers is dependent upon cellular cholesterol. Saffarian et al., using FCS, estimated that 70% of EGFRs exist as monomers, 20% as dimers, and 10% as oligomers (34). However, depletion of cholesterol decreases the percentage of monomeric receptors and increases the proportion of oligomeric receptors. Cholesterol depletion and actin depolymerization also alter the diffusion coefficient of the EGFR and the confinement area size (22). The finding that EGFR membrane organization is dependent upon cholesterol is of particular interest because a number of studies have demonstrated that EGFR activity is negatively regulated by cholesterol (4, 23, 28, 32).Phospholipase D2 (PLD2) hydrolyzes phosphatidylcholine (PC) to produce choline and phosphatidic acid (PA). PLD2 is localized to the plasma membrane (10), associates with the EGFR (39), and is rapidly activated upon EGF stimulation, leading to increased production of PA (15, 38, 39). A number of lines of evidence suggest that PA is an important mediator of EGFR action. First, exogenous PA is mitogenic when incubated with cells (17, 19, 42, 45). Second, direct interaction with membrane PA regulates the activity of a number of components downstream of the EGFR, including Sos (47) and Raf (12, 13, 30, 31).In the current study, we used high-resolution spatial analysis techniques to investigate EGFR plasma membrane organization. Using these approaches, we identified PA as the key molecular component responsible for driving EGFR nanocluster formation in response to EGF binding and demonstrated that the level of PLD2 activity regulates the duration of mitogen-activated protein kinase (MAPK) signal output.     

The First Transmembrane Domain of the Hepatitis B Virus Large Envelope Protein Is Crucial for Infectivity

The early steps of the hepatitis B virus (HBV) life cycle are still poorly understood. Indeed, neither the virus receptor at the cell surface nor the mechanism by which nucleocapsids are delivered to the cytosol of infected cells has been identified. Extensive mutagenesis studies in pre-S1, pre-S2, and most of the S domain of envelope proteins revealed the presence of two regions essential for HBV infectivity: the 77 first residues of the pre-S1 domain and a conformational motif in the antigenic loop of the S domain. In addition, at the N-terminal extremity of the S domain, a putative fusion peptide, partially overlapping the first transmembrane (TM1) domain and preceded by a PEST sequence likely containing several proteolytic cleavage sites, was identified. Since no mutational analysis of these two motifs potentially implicated in the fusion process was performed, we decided to investigate the ability of viruses bearing contiguous deletions or substitutions in the putative fusion peptide and PEST sequence to infect HepaRG cells. By introducing the mutations either in the L and M proteins or in the S protein, we demonstrated the following: (i) that in the TM1 domain of the L protein, three hydrophobic clusters of four residues were necessary for infectivity; (ii) that the same clusters were critical for S protein expression; and, finally, (iii) that the PEST sequence was dispensable for both assembly and infection processes.The hepatitis B virus (HBV) is the main human pathogen responsible for severe hepatic diseases like cirrhosis and hepatocellular carcinoma. Even though infection can be prevented by immunization with an efficient vaccine, about 2 billion people have been infected worldwide, resulting in 350 million chronic carriers that are prone to develop liver diseases (56). Current treatments consist either of the use of interferon α, which modulates antiviral defenses and controls infection in 30 to 40% of cases, or of the use of viral polymerase inhibitors that allow a stronger response to treatment but require long-term utilization and frequently lead to the outcome of resistant viruses (34, 55). A better understanding of the virus life cycle, and particularly of the mechanism by which the virus enters the cell, could provide background for therapeutics that inhibit the early steps of infection, as recently illustrated with the HBV pre-S1-derived entry inhibitor (25, 45).HBV belongs to the Hepadnaviridae family whose members infect different species. All viruses of this family share common properties. The capsid containing a partially double-stranded circular DNA genome is surrounded by a lipid envelope, in which two (in avihepadnaviruses infecting birds) or three (in orthohepadnaviruses infecting mammals) envelope proteins are embedded. A single open reading frame bearing several translation initiation sites encodes these surface proteins. Thus, the HBV envelope contains three proteins: S, M, and L that share the same C-terminal extremity corresponding to the small S protein that is crucial for virus assembly (7, 8, 46) and infectivity (1, 31, 53). These proteins are synthesized in the endoplasmic reticulum (ER), assembled, and secreted as particles through the Golgi apparatus (15, 42). The current model for the transmembrane structure of the S domain implies the luminal exposition of both N- and C-terminal extremities and the presence of four transmembrane (TM) domains: the TM1 and TM2 domains, both necessary for cotranslational protein integration into the ER membrane, and the TM3 and TM4 domains, located in the C-terminal third of the S domain (for a review, see reference 6). Among the four predicted TM domains, only the TM2 domain has a defined position between amino acids 80 and 98 of the S domain. The exact localization of the TM1 domain is still unclear, probably because of the relatively low hydrophobicity of its sequence, which contains polar residues and two prolines. The M protein corresponds to the S protein extended by an N-terminal domain of 55 amino acids called pre-S2. Its presence is dispensable for both assembly and infectivity (20, 21, 37). Finally, the L protein corresponds to the M protein extended by an N-terminal domain of 108 amino acids called pre-S1 (genotype D). The pre-S1 and pre-S2 domains of the L protein can be present either at the inner face of viral particles (on the cytoplasmic side of the ER), playing a crucial role in virus assembly (5, 8, 10, 11, 46), or on the outer face (on the luminal side of the ER), available for the interaction with target cells and necessary for viral infectivity (4, 14, 36). The pre-S translocation is independent from the M and S proteins and is driven by the L protein TM2 domain (33). Finally, HBV surface proteins are not only incorporated into virion envelopes but also spontaneously bud from ER-Golgi intermediate compartment membranes (30, 43) to form empty subviral particles (SVPs) that are released from the cell by secretion (8, 40).One approach to decipher viral entry is to interfere with the function of envelope proteins. Thus, by a mutagenesis approach, two envelope protein domains crucial for HBV infectivity have already been identified: (i) the 77 first amino acids of the pre-S1 domain (4, 36) including the myristic acid at the N-terminal extremity (9, 27) and (ii) possibly a cysteine motif in the luminal loop of the S domain (1, 31). In addition, a putative fusion peptide has been identified at the N-terminal extremity of the S domain due to its sequence homology with other viral fusion peptides (50). This sequence, either N-terminal in the S protein or internal in the L and M proteins, is conserved among the Hepadnaviridae family and shares common structural and functional properties with other fusion peptides (49, 50). Finally, a PEST sequence likely containing several proteolytic cleavage sites has been identified in the L and M proteins upstream of the TM1 domain (39). A cleavage within this sequence could activate the fusion peptide.In this study, we investigated whether the putative fusion peptide and the PEST sequence were necessary for the infection process. For this purpose, we constructed a set of mutant viruses bearing contiguous deletions in these regions and determined their infectivity using an in vitro infection model based on HepaRG cells (28). The introduction of mutations either in the L and M proteins or in only the S protein allowed us to demonstrate that, in the TM1 domain of L protein, three hydrophobic clusters not essential for viral assembly were crucial for HBV infectivity while their presence in the S protein was critical for envelope protein expression. In addition, we showed that the PEST sequence was clearly dispensable for both assembly and infection processes.     

HECT E3 Ubiquitin Ligase Nedd4-1 Ubiquitinates ACK and Regulates Epidermal Growth Factor (EGF)-Induced Degradation of EGF Receptor and ACK

ACK (activated Cdc42-associated tyrosine kinase) (also Tnk2) is an ubiquitin-binding protein and plays an important role in ligand-induced and ubiquitination-mediated degradation of epidermal growth factor receptor (EGFR). Here we report that ACK is ubiquitinated by HECT E3 ubiquitin ligase Nedd4-1 and degraded along with EGFR in response to EGF stimulation. ACK interacts with Nedd4-1 through a conserved PPXY WW-binding motif. The WW3 domain in Nedd4-1 is critical for binding to ACK. Although ACK binds to both Nedd4-1 and Nedd4-2 (also Nedd4L), Nedd4-1 is the E3 ubiquitin ligase for ubiquitination of ACK in cells. Interestingly, deletion of the sterile alpha motif (SAM) domain at the N terminus dramatically reduced the ubiquitination of ACK by Nedd4-1, while deletion of the Uba domain dramatically enhanced the ubiquitination. Use of proteasomal and lysosomal inhibitors demonstrated that EGF-induced ACK degradation is processed by lysosomes, not proteasomes. RNA interference (RNAi) knockdown of Nedd4-1, not Nedd4-2, inhibited degradation of both EGFR and ACK, and overexpression of ACK mutants that are deficient in either binding to or ubiquitination by Nedd4-1 blocked EGF-induced degradation of EGFR. Our findings suggest an essential role of Nedd4-1 in regulation of EGFR degradation through interaction with and ubiquitination of ACK.Activated Cdc42-associated tyrosine kinase (ACK) (also Tnk2) is a member of the type VIII tyrosine kinase family. Activation of ACK, including both ACK1 and ACK2, occurs in response to signaling of epidermal growth factor receptor (EGFR), platelet-derived growth factor (PDGF) receptor, insulin receptor, Gas-6 receptor (Mer), M3 muscarinic receptor, integrins, or proteoglycan (3, 7, 11, 23, 26, 30, 44, 47). In Drosophila, D-ACK mediates the function of Cdc42 in dorsal closure during embryonic development (31). The ACK homologue, Ark-1, in Caenorhabditis elegans negatively regulates EGF signaling (15).A number of studies suggest a role for ACK in EGFR degradation. ACK1 and ACK2, two alternatively spliced isoforms, possess a highly conserved clathrin-binding motif and interact with clathrin (37, 45). Overexpression of ACK2 severely impairs transferrin receptor endocytosis, causes aberrant localization of AP-2, and induces changes in clathrin assembly. Furthermore, ACK2 interacts with sorting nexin 9 (SNX9, also named SH3PX1), a member of the sorting nexin family, via its proline-rich domain 1 and phosphorylates SNX9 to facilitate the degradation of EGF receptors (22). In C. elegans, Ark-1 genetically interacts with UNC101, the homologue of mammalian clathrin-associated protein AP47, and SLI-1, the homologue of mammalian Cbl that is an E3 ubiquitin ligase for ubiquitination of EGFR, and negatively regulates EGFR signaling (15).Our previous studies showed that ACK1 interacts with EGFR upon EGF stimulation via a region at the carboxyl terminus, designated the EGFR-binding domain (EBD), which is highly homologous to the EGFR/ErbB2-binding domain of Gene-33/Mig-6/RALT (32, 43). The interaction of ACK1 with EGFR is dependent on kinase activity and tyrosine phosphorylation of EGFR. Immunofluorescent staining using anti-EGFR and GFP-ACK1 indicates that ACK1 is colocalized with EGFR on large vacuolar structures upon EGF stimulation. Suppression of the expression of ACK1 by ACK-RNA interference (RNAi) inhibits ligand-induced degradation of EGFR, suggesting that ACK1 plays an important role in the regulation of EGFR degradation in cells. Furthermore, we identified ACK1 as an ubiquitin-binding protein. Through an ubiquitin association (Uba) domain at the carboxyl terminus, ACK1 is capable of interacting with both poly- and monoubiquitin. Overexpression of an Uba domain deletion mutant of ACK1 blocked the ligand-dependent degradation of EGFR, suggesting that ACK1 regulates EGFR degradation via its Uba domain. Thus, ACK1 senses EGF signaling and regulates degradation of EGFR.EGF-induced degradation of EGFR is mediated by ubiquitination (16). The ubiquitination of EGFR is activated upon EGF stimulation by recruiting the RING family E3 ubiquitin ligase Cbl to pY1045 (20, 21). This ubiquitination functions as a sorting signal for transporting EGFR to lysosomes for degradation (14). Nedd4, the HECT domain-containing E3 ubiquitin ligase, is also involved in the regulation of EGFR trafficking by ubiquitination of endocytic or vesicle sorting proteins (28). For example, it has been observed that Nedd4 ubiquitinates Cbl, Eps15, Tsg101, Hrs, and secretory carrier membrane proteins (SCAMPs) and participates in the processes of EGFR endocytosis and degradation (1, 18, 25, 42). However, exactly how Nedd4 engages in the EGFR degradation process in response to EGF stimulation is not known.In this report, we show that EGF stimulation induces ACK degradation. This degradation is associated with ubiquitination of ACK. Nedd4-1, but not Nedd4-2, is identified as the E3 ubiquitin ligase for ubiquitination of ACK. Furthermore, EGF-induced degradation of ACK is EGFR activation dependent and processed by lysosomes. RNAi knockdown and mutational analysis demonstrated that Nedd4-1 and Nedd4-1-catalyzed ubiquitination of ACK are required for EGF-induced degradation of EGFR and ACK. Our findings suggest a new mechanism in regulation of EGFR degradation.     

Protein Tyrosine Kinase 6 Directly Phosphorylates AKT and Promotes AKT Activation in Response to Epidermal Growth Factor

Protein tyrosine kinase 6 (PTK6) is a nonmyristoylated Src-related intracellular tyrosine kinase. Although not expressed in the normal mammary gland, PTK6 is expressed in a majority of human breast tumors examined, and it has been linked to ErbB receptor signaling and AKT activation. Here we demonstrate that AKT is a direct substrate of PTK6 and that AKT tyrosine residues 315 and 326 are phosphorylated by PTK6. Association of PTK6 with AKT occurs through the SH3 domain of PTK6 and is enhanced through SH2 domain-mediated interactions following tyrosine phosphorylation of AKT. Using Src, Yes, and Fyn null mouse embryonic fibroblasts (SYF cells), we show that PTK6 phosphorylates AKT in a Src family kinase-independent manner. Introduction of PTK6 into SYF cells sensitized these cells to physiological levels of epidermal growth factor (EGF) and increased AKT activation. Stable introduction of active PTK6 into SYF cells also resulted in increased proliferation. Knockdown of PTK6 in the BPH-1 human prostate epithelial cell line led to decreased AKT activation in response to EGF. Our data indicate that in addition to promoting growth factor receptor-mediated activation of AKT, PTK6 can directly activate AKT to promote oncogenic signaling.Protein tyrosine kinase 6 (PTK6; also known as the breast tumor kinase BRK) is an intracellular Src-related tyrosine kinase (9, 48). Human PTK6 was identified in cultured human melanocytes (32) and breast tumor cells (39), while its mouse orthologue was cloned from normal small intestinal epithelial cell RNA (50). Although PTK6 shares overall structural similarity with Src family tyrosine kinases, it lacks an N-terminal myristoylation consensus sequence for membrane targeting (39, 51). As a consequence, PTK6 is localized to different cellular compartments, including the nucleus (14, 15). PTK6 is expressed in normal differentiated epithelial cells of the gastrointestinal tract (34, 42, 51), prostate (14), and skin (51-53). Expression of PTK6 is upregulated in different types of cancers, including breast carcinomas (6, 39, 54), colon cancer (34), ovarian cancer (47), head and neck cancers (33), and metastatic melanoma cells (16). The significance of apparent opposing signaling roles for PTK6 in normal differentiation and cancer is still poorly understood.In human breast tumor cells, PTK6 enhances signaling from members of the ErbB receptor family (10, 29, 30, 36, 40, 49, 54). In the HB4a immortalized human mammary gland luminal epithelial cell line, PTK6 promoted epidermal growth factor (EGF)-induced ErbB3 tyrosine phosphorylation and AKT activation (29). In response to EGF stimulation, PTK6 promoted phosphorylation of the focal adhesion protein paxillin and Rac1-mediated cell migration (10). PTK6 can be activated by the ErbB3 ligand heregulin and promotes activation of extracellular signal-regulated kinase 5 (ERK5) and p38 mitogen-activated protein kinase (MAPK) in breast cancer cells (40). PTK6 can also phosphorylate p190RhoGAP-A and stimulate its activity, leading to RhoA inactivation and Ras activation and thereby promoting EGF-dependent breast cancer cell migration and proliferation (49). Expression of PTK6 has been correlated with ErbB2 expression in human breast cancers (4, 5, 54).AKT (also called protein kinase B) is a serine-threonine kinase that is activated downstream of growth factor receptors (38). It is a key player in signaling pathways that regulate energy metabolism, proliferation, and cell survival (7, 45). Aberrant activation of AKT through diverse mechanisms has been discovered in different cancers (2). AKT activation requires phosphorylation of AKT on threonine residue 308 and serine residue 473. The significance of phosphorylation of AKT on tyrosine residues is less well understood. Src has been shown to phosphorylate AKT on conserved tyrosine residues 315 and 326 near the activation loop (11). Substitution of these two tyrosine residues with phenylalanine abolished AKT kinase activity stimulated by EGF (11). Use of the Src family inhibitor PP2 impaired AKT activation following IGF-1 stimulation of oligodendrocytes (13). The RET/PTC receptor tyrosine kinase that responds to glial cell-line-derived neurotrophic factor also phosphorylated AKT tyrosine residue 315 promoting activation of AKT (28). AKT tyrosine residue 474 was phosphorylated when cells were treated with the tyrosine phosphatase inhibitor pervanadate, and phosphorylation of tyrosine 474 contributed to full activation of AKT (12). Recently, the nonreceptor tyrosine kinase Ack1 was shown to regulate AKT tyrosine phosphorylation and activation (37).Here we show that AKT is a cytoplasmic substrate of the intracellular tyrosine kinase PTK6. We identify the tyrosine residues on AKT that are targeted by PTK6, and we demonstrate that tyrosine phosphorylation plays a role in regulating association between PTK6 and AKT. In addition, we show that PTK6 promotes AKT activation and cell proliferation in a Src-independent manner.     

Ebola Virus Glycoprotein Counteracts BST-2/Tetherin Restriction in a Sequence-Independent Manner That Does Not Require Tetherin Surface Removal

BST-2/tetherin is an interferon-inducible protein that restricts the release of enveloped viruses from the surface of infected cells by physically linking viral and cellular membranes. It is present at both the cell surface and in a perinuclear region, and viral anti-tetherin factors including HIV-1 Vpu and HIV-2 Env have been shown to decrease the cell surface population. To map the domains of human tetherin necessary for both virus restriction and sensitivity to viral anti-tetherin factors, we constructed a series of tetherin derivatives and assayed their activity. We found that the cytoplasmic tail (CT) and transmembrane (TM) domains of tetherin alone produced its characteristic cellular distribution, while the ectodomain of the protein, which includes a glycosylphosphatidylinositol (GPI) anchor, was sufficient to restrict virus release when presented by the CT/TM regions of a different type II membrane protein. To counteract tetherin restriction and remove it from the cell surface, HIV-1 Vpu required the specific sequence present in the TM domain of human tetherin. In contrast, the HIV-2 Env required only the ectodomain of the protein and was sensitive to a point mutation in this region. Strikingly, the anti-tetherin factor, Ebola virus GP, was able to overcome restriction conferred by both tetherin and a series of functional tetherin derivatives, including a wholly artificial tetherin molecule. Moreover, GP overcame restriction without significantly removing tetherin from the cell surface. These findings suggest that Ebola virus GP uses a novel mechanism to circumvent tetherin restriction.Pathogenic viruses often have evolved mechanisms to neutralize host defenses that act at the cellular level to interfere with the virus life cycle. Such cellular restriction factors have been most extensively characterized for HIV-1 (38) and include the interferon-inducible membrane protein BST-2/HM1.24/CD317/tetherin (28, 40). If unchecked, tetherin blocks the release of newly formed HIV-1 particles from cells by physically tethering them at the cell surface (7, 28, 32, 40). In addition, tetherin has been shown to act against a broad range of enveloped viral particles, including retroviruses, filoviruses, arenaviruses, and herpesviruses (17, 18, 23, 35). In turn, certain viruses that are targeted by tetherin appear to have evolved counteracting activities, and anti-tetherin factors so far identified include HIV-1 Vpu; HIV-2 Env; simian immunodeficiency virus (SIV) Nef, Vpu, and Env proteins; Ebola virus GP; and Kaposi''s sarcoma-associated herpesvirus (KSHV) K5 (11, 16, 18, 20, 23, 28, 36, 40, 44, 45).Tetherin is a homodimeric type II integral membrane protein containing an N-terminal cytoplasmic tail (CT), a single-pass transmembrane domain (TM), an ectodomain-containing predicted coiled-coil regions, two glycoslyation sites, three conserved cysteines, and a C-terminal glycosylphosphatidylinositol (GPI) anchor (2, 19, 31). This unusual topology, with two independent membrane anchors, has led to the suggestion that the retention of virions at the cell surface arises from tetherin''s ability to be inserted simultaneously in both host and viral membranes (28, 32, 41) or, alternatively, that dimers or higher-order complexes of tetherin conferred by the ectodomain mediate this effect (39). Interestingly, an artificial tetherin containing the same structural features as the native protein but constructed from unrelated sequences was able to restrict both HIV-1 and Ebola virus particles (32). This suggests that the viral lipid envelope is the target of tetherin and provides an explanation for tetherin''s broad activity against diverse enveloped viruses.A fraction of tetherin is present at the plasma membrane of cells (9, 14), and it has been proposed that viral anti-tetherin factors function by removing this cell surface fraction (40). This now has been shown to occur in the presence of HIV-1 Vpu (5, 7, 15, 26, 34, 40, 44), HIV-2 Env (5, 20), SIV Env (11), SIV Nef (15), and KSHV K5 (3, 23). In addition, certain anti-tetherin factors also may promote the degradation of tetherin, as has been observed for both HIV-1 Vpu (3, 5, 7, 10, 22, 26, 27) and KSHV K5 (3, 23), although Vpu also appears able to block tetherin restriction in the absence of degradation (8), and no effects on tetherin steady-state levels have been observed in the presence of either the HIV-2 or SIVtan Env (11, 20). Simply keeping tetherin away from the cell surface, or targeting it for degradation, may not be the only mechanism used by anti-tetherin factors, since it also has been reported that Vpu does not affect the levels of surface tetherin or its total cellular levels in certain T-cell lines (27).The interactions between tetherin and viral anti-tetherin factors show evidence of species specificity, suggesting ongoing evolution between viruses and their hosts. HIV-1 Vpu is active against human and chimpanzee tetherin but not other primate tetherins (10, 25, 34, 36, 44, 45), while SIV Nef proteins are active against primate but not human tetherins (16, 36, 44, 45). This suggests that, unlike tetherin restriction, the action of the anti-tetherin factors may involve specific sequence interactions. Indeed, the TM domain has been recognized as a target for HIV-1 Vpu (10, 15, 16, 25, 34), while a single point mutation introduced into the extracellular domain of human tetherin can block its antagonism by the SIVtan Env (11).In the present study, we investigated the roles of the different domains of tetherin in both promoting virus restriction and conferring susceptibility to the anti-tetherin factors encoded by HIV-1, HIV-2, and Ebola virus. We confirmed that tetherin restriction can be conferred by proteins that retain the two distinct membrane anchors, while signals for the cellular localization of the protein reside in the CT/TM domains of the protein. We found that the Vpu protein targets the TM domain of tetherin, while the HIV-2 Env targets the ectodomain of the protein. In contrast, the Ebola virus GP appears to use a non-sequence-specific mechanism to counteract tetherin restriction, since even an artificial tetherin could be successfully overcome by GP expression. Interestingly, Ebola virus GP counteracted tetherin restriction without removing the protein from the cell surface, suggesting that it is possible to overcome this restriction by mechanisms other than blocking tetherin''s cell surface expression.     

Residues in a Highly Conserved Claudin-1 Motif Are Required for Hepatitis C Virus Entry and Mediate the Formation of Cell-Cell Contacts

Claudin-1, a component of tight junctions between liver hepatocytes, is a hepatitis C virus (HCV) late-stage entry cofactor. To investigate the structural and functional roles of various claudin-1 domains in HCV entry, we applied a mutagenesis strategy. Putative functional intracellular claudin-1 domains were not important. However, we identified seven novel residues in the first extracellular loop that are critical for entry of HCV isolates drawn from six different subtypes. Most of the critical residues belong to the highly conserved claudin motif W₃₀-GLW₅₁-C₅₄-C₆₄. Alanine substitutions of these residues did not impair claudin-1 cell surface expression or lateral protein interactions within the plasma membrane, including claudin-1-claudin-1 and claudin-1-CD81 interactions. However, these mutants no longer localized to cell-cell contacts. Based on our observations, we propose that cell-cell contacts formed by claudin-1 may generate specialized membrane domains that are amenable to HCV entry.Hepatitis C virus (HCV) is a major human pathogen that affects approximately 3% of the global population, leading to cirrhosis and hepatocellular carcinoma in chronically infected individuals (5, 23, 42). Hepatocytes are the major target cells of HCV (11), and entry follows a complex cascade of interactions with several cellular factors (6, 8, 12, 17). Infectious viral particles are associated with lipoproteins and initially attach to target cells via glycosaminoglycans and the low-density lipoprotein receptor (1, 7, 31). These interactions are followed by direct binding of the E2 envelope glycoprotein to the scavenger receptor class B type I (SR-B1) and then to the CD81 tetraspanin (14, 15, 33, 36). Early studies showed that CD81 and SR-B1 were necessary but not sufficient for HCV entry, and claudin-1 was discovered to be a requisite HCV entry cofactor that appears to act at a very late stage of the process (18).Claudin-1 is a member of the claudin protein family that participates in the formation of tight junctions between adjacent cells (25, 30, 37). Tight junctions regulate the paracellular transport of solutes, water, and ions and also generate apical-basal cell polarity (25, 37). In the liver, the apical surfaces of hepatocytes form bile canaliculi, whereas the basolateral surfaces face the underside of the endothelial layer that lines liver sinusoids. Claudin-1 is highly expressed in tight junctions formed by liver hepatocytes as well as on all hepatoma cell lines that are permissive to HCV entry (18, 24, 28). Importantly, nonhepatic cell lines that are engineered to express claudin-1 become permissive to HCV entry (18). Claudin-6 and -9 are two other members of the human claudin family that enable HCV entry into nonpermissive cells (28, 43).The precise role of claudin-1 in HCV entry remains to be determined. A direct interaction between claudins and HCV particles or soluble E2 envelope glycoprotein has not been demonstrated (18; T. Dragic, unpublished data). It is possible that claudin-1 interacts with HCV entry receptors SR-B1 or CD81, thereby modulating their ability to bind to E2. Alternatively, claudin-1 may ferry the receptor-virus complex to fusion-permissive intracellular compartments. Recent studies show that claudin-1 colocalizes with the CD81 tetraspanin at the cell surface of permissive cell lines (22, 34, 41). With respect to nonpermissive cells, one group observed that claudin-1 was predominantly intracellular (41), whereas another reported associations of claudin-1 and CD81 at the cell surface, similar to what is observed in permissive cells (22).Claudins comprise four transmembrane domains along with two extracellular loops and two cytoplasmic domains (19, 20, 25, 30, 37). The first extracellular loop (ECL1) participates in pore formation and influences paracellular charge selectivity (25, 37). It has been shown that the ECL1 of claudin-1 is required for HCV entry (18). All human claudins comprise a highly conserved motif, W₃₀-GLW₅₁-C₅₄-C₆₄, in the crown of ECL1 (25, 37). The exact function of this domain is unknown, and we hypothesized that it is important for HCV entry. The second extracellular loop is required for the holding function and oligomerization of the protein (25). Claudin-1 also comprises various signaling domains and a PDZ binding motif in the intracellular C terminus that binds ZO-1, another major component of tight junctions (30, 32, 37). We further hypothesized that some of these domains may play a role in HCV entry.To understand the role of claudin-1 in HCV infection, we developed a mutagenesis strategy targeting the putative sites for internalization, glycosylation, palmitoylation, and phosphorylation. The functionality of these domains has been described by others (4, 16, 25, 35, 37, 40). We also mutagenized charged and bulky residues in ECL1, including all six residues within the highly conserved motif W₃₀-GLW₅₁-C₅₄-C₆₄. None of the intracellular domains were found to affect HCV entry. However, we identified seven residues in ECL1 that are critical for entry mediated by envelope glycoproteins derived from several HCV subtypes, including all six residues of the conserved motif. These mutants were still expressed at the cell surface and able to form lateral homophilic interactions within the plasma membrane as well as to engage in lateral interactions with CD81. In contrast, they no longer engaged in homophilic trans interactions at cell-cell contacts. We conclude that the highly conserved motif W₃₀-GLW₅₁-C₅₄-C₆₄ of claudin-1 is important for HCV entry into target cells and participates in the formation of cell-cell contacts.     

Divergent Bro1 Domains Share the Capacity To Bind Human Immunodeficiency Virus Type 1 Nucleocapsid and To Enhance Virus-Like Particle Production

To promote the release of infectious virions, human immunodeficiency virus type 1 (HIV-1) exploits the endosomal sorting complex required for transport (ESCRT) pathway by engaging Tsg101 and ALIX through late assembly (L) domains in p6 Gag. An LYPx_nL motif in p6 serves as docking site for the central V domain of ALIX and is required for its ability to stimulate HIV-1 budding. Additionally, the nucleocapsid (NC) domain of Gag binds to the N-terminal Bro1 domain of ALIX, which connects ALIX to the membrane-deforming ESCRT-III complex via its CHMP4 subunits. Since the isolated Bro1 domain of ALIX is sufficient to markedly stimulate virus-like particle (VLP) production in a minimal Gag rescue assay, we examined whether the Bro1 domains of other human proteins possess a similar activity. We now show that the Bro1 domain-only protein Brox and the isolated Bro1 domains of HD-PTP and rhophilin all bind to HIV-1 NC. Furthermore, all shared the capacity to stimulate VLP production by a minimal HIV-1 Gag molecule, and Brox in particular was as potent as the Bro1 domain of ALIX in this assay. Unexpectedly, Brox retained significant activity even if its CHMP4 binding site was disrupted. Thus, the ability to assist in VLP production may be an intrinsic property of the boomerang-shaped Bro1 domain.Retroviruses engage an endosomal budding machinery via so-called late assembly (L) domains in Gag to promote virus budding at the plasma membrane (4, 17, 33). In the case of human immunodeficiency virus type 1 (HIV-1), the C-terminal p6 domain of Gag harbors a conserved P(T/S)AP motif, which binds to the host protein Tsg101 and functions as the primary L domain (18, 29, 44). Additionally, HIV-1 p6 contains an auxiliary L domain of the LYPx_nL type, which serves as a docking site for ALIX (28, 41, 45). Tsg101 and ALIX are both components of a protein network that is required for the biogenesis of multivesicular bodies (MVB) (22, 38). These compartments are formed through the budding of vesicles from the limiting membrane of endosomes into their lumen, a process that is topologically equivalent to virus budding at the plasma membrane. Recently, it emerged that the protein network essential for MVB formation also functions in cytokinesis, which requires a membrane fission event of similar topology (7, 32).Most of the components of the protein network that mediates these events are subunits of heteromeric endosomal sorting complexes required for transport (ESCRT) (3, 22, 38). For instance, Tsg101 is a subunit of the heterotetrameric ESCRT-I complex (22, 38). ESCRT-I and the downstream ESCRT-II are stable complexes, whereas ESCRT-III assembles only upon membrane binding (38). ESCRT-III is formed by the structurally related human CHMP proteins, which exist in an autoinhibited monomeric conformation in the cytosol (40, 46). A conformational change from a closed to an open conformation is thus likely required for the activation of CHMP proteins and the assembly of ESCRT-III. Interestingly, the uncontrolled activation of CHMP proteins through the removal of autoinhibitory C-terminal sequences results in the potent inhibition of HIV-1 budding, indicating a central role for ESCRT-III in retroviral release (46).ALIX consists of a boomerang-shaped N-terminal Bro1 domain, a central ligand binding domain that is shaped like a V, and a C-terminal proline-rich region (16). While ALIX is essential for equine anemia virus budding, its role in HIV-1 budding is less critical than that of Tsg101 (8, 16, 28, 41). However, ALIX can clearly support efficient HIV-1 budding, because its overexpression potently rescues the release defect of Tsg101 binding site mutants (16, 43). This effect of ALIX depends on the interaction between its central V domain and the LYPx_nL motif in HIV-1 p6 (16, 43), confirming that this motif constitutes a functional L domain.The Bro1 domain of ALIX interacts tightly with ESCRT-III subunit CHMP4B and less avidly with CHMP4A and CHMP4C (25, 28, 41, 45). The ability of ALIX to rescue HIV-1 L domain mutants depends on the interaction between its Bro1 domain and CHMP4, indicating that CHMP4 is of particular importance in viral budding (16, 43). Interestingly, human CHMP4A assembles into membrane-attached filaments if overexpressed in mammalian cells, and these filaments can be induced to form circular arrays that drive the formation of buds and tubules with the same topology as that of a retroviral bud (21). Also, the single yeast ortholog of the mammalian CHMP4 proteins forms homo-oligomeric filaments on endosomes that appear to drive MVB sorting and biogenesis (42).By binding to membranes with its convex surface, the Bro1 domain of ALIX could also contribute directly to the generation of negative curvature required for budding away from the cytosol. In support of this notion, we recently observed that the isolated Bro1 domain of ALIX can potently enhance the formation of virus-like particles (VLP) by a minimal HIV-1 Gag construct that retains the primary L domain but lacks certain assembly domains and thus is presumably defective in its ability to deform membranes (37). We also observed that the Bro1 domain of ALIX physically interacts with the nucleocapsid (NC) region of HIV-1 Gag and that mutations in NC that interfere with the interaction induce a phenotype that resembles that of L domain mutants (37).Despite limited sequence homology between human ALIX and a yeast counterpart, the structures of their Bro1 domains are largely superimposable (16, 26), suggesting that all Bro1 domains have a shape that would be compatible with a membrane-deforming function. We therefore asked whether the ability to stimulate VLP production is unique to the Bro1 domain of ALIX or a property of Bro1 domains in general. We now show that widely divergent Bro1 domains share the ability to associate with HIV-1 Gag in an NC-dependent manner and to enhance VLP production by a minimal Gag molecule. In particular, a human Bro1 domain-only protein termed Brox (23) was as potent as the ALIX Bro1 domain in stimulating VLP production, and even forms of Brox that did not bind to CHMP4 retained significant activity. We thus propose that Bro1 domains are inherently capable of promoting budding events away from the cytosol.     

Human Immunodeficiency Virus Type 1 Nucleocapsid p1 Confers ESCRT Pathway Dependence

To facilitate the release of infectious progeny virions, human immunodeficiency virus type 1 (HIV-1) exploits the Endosomal Sorting Complex Required for Transport (ESCRT) pathway by engaging Tsg101 and ALIX through late assembly (L) domains in the C-terminal p6 domain of Gag. However, the L domains in p6 are known to be dispensable for efficient particle production by certain HIV-1 Gag constructs that have the nucleocapsid (NC) domain replaced by a foreign dimerization domain to substitute for the assembly function of NC. We now show that one such L domain-independent HIV-1 Gag construct (termed Z_WT) that has NC-p1-p6 replaced by a leucine zipper domain is resistant to dominant-negative inhibitors of the ESCRT pathway that block HIV-1 particle production. However, Z_WT became dependent on the presence of an L domain when NC-p1-p6 was restored to its C terminus. Furthermore, when the NC domain was replaced by a leucine zipper, the p1-p6 region, but not p6 alone, conferred sensitivity to inhibition of the ESCRT pathway. In an authentic HIV-1 Gag context, the effect of an inhibitor of the ESCRT pathway on particle production could be alleviated by deleting a portion of the NC domain together with p1. Together, these results indicate that the ESCRT pathway dependence of HIV-1 budding is determined, at least in part, by the NC-p1 region of Gag.Human immunodeficiency virus type 1 (HIV-1) and other retroviruses hijack the cellular Endosomal Sorting Complex Required for Transport (ESCRT) pathway to promote the detachment of virions from the cell surface and from each other (3, 21, 42, 44, 47). The ESCRT pathway was initially identified based on its requirement for the sorting of ubiquitinated cargo into multivesicular bodies (MVB) (50, 51). During MVB biogenesis, the ESCRT pathway drives the membrane deformation and fission events required for the inward vesiculation of the limiting membrane of this organelle (26, 29, 50, 51). More recently, it emerged that the ESCRT pathway is also essential for the normal abscission of daughter cells during the final stage of cell division (10, 43). Most of the components of the ESCRT pathway are involved in the formation of four heteromeric protein complexes termed ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III. Additional components include ALIX, which interacts both with ESCRT-I and ESCRT-III, and the AAA ATPase Vps4, which mediates the disassembly of ESCRT-III (29, 42).The deformation and scission of endocytic membranes is thought to be mediated by ESCRT-III, which, together with Vps4, constitutes the most conserved element of the pathway (23, 26, 42). Indeed, it was recently shown that purified yeast ESCRT-III induces membrane deformation (52), and in another study three subunits of yeast ESCRT-III were sufficient to promote the formation of intralumenal vesicles in an in vitro assay (61). In mammals, ESCRT-III is formed by the charged MVB proteins (CHMPs), which are structurally related and tightly regulated through autoinhibition (2, 33, 46, 53, 62). The removal of an inhibitory C-terminal domain induces polymerization and association with endosomal membranes and converts CHMPs into potent inhibitors of retroviral budding (34, 46, 53, 60, 62). Alternatively, CHMPs can be converted into strong inhibitors of the ESCRT pathway and of HIV-1 budding through the addition of a bulky tag such as green fluorescent protein (GFP) or red fluorescent protein (RFP) (27, 36, 39, 54). Retroviral budding in general is also strongly inhibited by catalytically inactive Vps4 (22, 41, 55), or upon Vsp4B depletion (31), confirming the crucial role of ESCRT-III.Retroviruses engage the ESCRT pathway through the activity of so-called late assembly (L) domains in Gag. In the case of HIV-1, the primary L domain maps to a conserved PTAP motif in the C-terminal p6 domain of Gag (24, 28) and interacts with the ESCRT-I component Tsg101 (15, 22, 40, 58). HIV-1 p6 also harbors an auxiliary L domain of the LYPx_nL type, which interacts with the V domain of ALIX (20, 35, 39, 54, 59, 63). Interestingly, Tsg101 binding site mutants of HIV-1 can be fully rescued through the overexpression of ALIX, and this rescue depends on the ALIX binding site in p6 (20, 56). In contrast, the overexpression of a specific splice variant of the ubiquitin ligase Nedd4-2 has been shown to rescue the release and infectivity of HIV-1 mutants lacking all known L domains in p6 (12, 57). Nedd4 family ubiquitin ligases had previously been implicated in the function of PPxY-type L domains, which also depend on an intact ESCRT pathway for function (4, 32, 38). However, HIV-1 Gag lacks PPxY motifs, and the WW domains of Nedd4-2, which mediate its interaction with PPxY motifs, are dispensable for the rescue of HIV-1 L domain mutants (57).ALIX also interacts with the nucleocapsid (NC) region of HIV-1 Gag (18, 49), which is located upstream of p6 and the p1 spacer peptide. ALIX binds HIV-1 NC via its Bro1 domain, and the capacity to interact with NC and to stimulate the release of a minimal HIV-1 Gag construct is shared among widely divergent Bro1 domain proteins (48). Based on these findings and the observation that certain mutations in NC cause a phenotype that resembles that of L domain mutants, it has been proposed that NC cooperates with p6 to recruit the machinery required for normal HIV-1 budding (18, 49).NC also plays a role in Gag polyprotein multimerization, and this function of NC depends on its RNA-binding activity (5-8). It has been proposed that the role of the NC-nucleic acid interaction during assembly is to promote the formation of Gag dimers (37), and HIV-1 assembly in the absence of NC can indeed be efficiently rescued by leucine zipper dimerization domains (65). Surprisingly, in this setting the L domains in p6 also became dispensable, since particle production remained efficient even when the entire NC-p1-p6 region of HIV-1 Gag was replaced by a leucine zipper (1, 65). These findings raised the possibility that the reliance of wild-type (WT) HIV-1 Gag on a functional ESCRT pathway is, at least in part, specified by NC-p1-p6. However, it also remained possible that the chimeric Gag constructs engaged the ESCRT pathway in an alternative manner.In the present report, we provide evidence supporting the first of those two possibilities. Particle production became independent of ESCRT when the entire NC-p1-p6 region was replaced by a leucine zipper, and reversion to ESCRT dependence was shown to occur as a result of restoration of p1-p6 but not of p6 alone. Furthermore, although the deletion of p1 alone had little effect in an authentic HIV-1 Gag context, the additional removal of a portion of NC improved particle production in the presence of an inhibitor of the ESCRT pathway. Together, these data imply that the NC-p1 region plays an important role in the ESCRT-dependence of HIV-1 particle production.     

Regulation of IRSp53-Dependent Filopodial Dynamics by Antagonism between 14-3-3 Binding and SH3-Mediated Localization

Filopodia are dynamic structures found at the leading edges of most migrating cells. IRSp53 plays a role in filopodium dynamics by coupling actin elongation with membrane protrusion. IRSp53 is a Cdc42 effector protein that contains an N-terminal inverse-BAR (Bin-amphipysin-Rvs) domain (IRSp53/MIM homology domain [IMD]) and an internal SH3 domain that associates with actin regulatory proteins, including Eps8. We demonstrate that the SH3 domain functions to localize IRSp53 to lamellipodia and that IRSp53 mutated in its SH3 domain fails to induce filopodia. Through SH3 domain-swapping experiments, we show that the related IRTKS SH3 domain is not functional in lamellipodial localization. IRSp53 binds to 14-3-3 after phosphorylation in a region that lies between the CRIB and SH3 domains. This association inhibits binding of the IRSp53 SH3 domain to proteins such as WAVE2 and Eps8 and also prevents Cdc42-GTP interaction. The antagonism is achieved by phosphorylation of two related 14-3-3 binding sites at T340 and T360. In the absence of phosphorylation at these sites, filopodium lifetimes in cells expressing exogenous IRSp53 are extended. Our work does not conform to current views that the inverse-BAR domain or Cdc42 controls IRSp53 localization but provides an alternative model of how IRSp53 is recruited (and released) to carry out its functions at lamellipodia and filopodia.The ability of a cell to rapidly respond to extracellular cues and direct cytoskeletal rearrangements is dependent on an array of signaling complexes that control actin assembly (58). The protrusive structures at the leading edges of motile cells are broadly defined as lamellipodia or filopodia (14). Lamellae are sheet-like protrusions composed of dendritic actin arrays that drive membrane expansion, with the “lamellipodium” representing a narrow region at the edge of the cell (in culture) characterized by rapid actin polymerization. This F-actin assembly is suggested to require Arp2/3 activity that nucleates new actin filaments from the sides of existing ones (58, 71) and capping proteins that limit the length of these new filaments and stabilize them (7). Arp2/3 activity in turn is regulated by the WASP/WAVE family of proteins, such as N-WASP and WAVE2 (68), whose regulation is a subject of intense interest (12, 29, 36, 41, 56, 76).Filopodia contain parallel bundles of actin filaments containing fascin (22). These are dynamic structures that emanate from the periphery of the cell and are retracted, with occasional attachment (to the dish in culture). Thus, they have been thought to have a sensory or exploratory role during cell migration (28). This is the case for neuronal growth cones, where filopodia sense attractant or repulsive cues and dictate direction in axonal path finding (9, 17, 25, 35). Filopodia have been shown to be important in the context of dendritic-spine development (64, 77), epithelial-sheet closure (26, 60, 79), and cell invasion/metastasis (80, 83).Lamellipodia have been well characterized since the pioneering work of Abercrombie et al. in the early 1970s (2, 3, 4). Filopodia require symmetry breaking at the leading edge (initiation), followed by elongation driven by a filopodial-tip protein complex (14, 28). A few proteins have been identified in this complex; Mena/Vasp serve to prevent capping at the barbed ends of bundled actin filaments (7, 53), and Dia2 promotes F-actin elongation (57, 85). Termination of filopodial elongation is not understood but nonetheless is likely to be tightly regulated. In the absence of F-actin elongation, retraction of the filopodium takes place by a rearward flow of F-actin and filament depolymerization (22).IRSp53 is in a position to play a pivotal role in generating filopodia; this brain-enriched protein was discovered as a substrate of the insulin receptor (87). Subsequently, IRSp53 was identified as an effector for Rac1 (50) and Cdc42 (27, 38), where it participates in filopodium and lamellipodium production (38, 51, 54, 86), neurite extension (27), dendritic-spine morphogenesis (1, 15, 66, 67), cell motility and invasiveness (24). The N terminus of IRSp53 contains a conserved helical domain that is found in five different gene products and is referred to as the IRSp53/MIM homology domain (IMD) (51, 70). This domain has been postulated to bind to Rac1 (50, 70) in a nucleotide-independent manner (52), but no convincing effector-like region has been identified. A Cdc42-specific CRIB-like sequence that does not bind Rac1 (27, 38) allows coupling of this and perhaps related Rho GTPases. The structure of the IMD reveals a zeppelin-shaped dimer that could bind “bent” membranes; thus, its potential as an F-actin-bundling domain (51, 82) could be an in vitro artifact often attributed to proteins with basic patches (46). Although there are reports of F-actin binding at physiological ionic strength (ca. 100 mM KCl) (82, 19), this region when expressed in isolation does not decorate F-actin in vivo.Two reports showed the IMD to be an “inverse-BAR” domain. BAR (Bin-amphipysin-Rvs) domains are found in proteins involved in endocytic trafficking, such as amphipysin and endophilin, and stabilize positively bent membranes, such as those on endocytic vesicles (31, 47). The IMD domains of both IRSp53 (70) and MIM-B (46) associate with lipids and can induce tubulations of PI(3,4,5)P₃ or PI(4,5)P₂-rich membranes, respectively. These tubulations are equivalent to membrane protrusions and are also referred to as negatively bent membranes. Ectopic expression of the IMD from IRSp53 (51, 70, 82, 86) or two other family members, MIM-B (11, 46) and IRTKS (52), can give rise to cells with many peripheral extensions. MIM-B is said to stimulate lamellipodia (11), while IRTKS generates “short actin clusters” at the cell periphery (52).In IRSp53 is a CRIB-like motif that mediates binding to Cdc42 (27, 38), but the function of this interaction in unclear. Cdc42 could relieve IRSp53 autoinhibition as described for N-Wasp (38), but there is little evidence for this. It has been suggested that Cdc42 controls IRSp53 localization and actin remodeling (27, 38), but another study indicated that these events are Cdc42 independent (19). IRSp53 contains a central SH3 domain that may bind proline-rich proteins, such as Dia1 (23), Mena (38), WAVE2 (49, 50, 69), and Eps8 (19, 24). However, it seems unlikely that all of these represent bona fide partners, and side-by-side comparison is provided in this study. Mena is involved in filopodium production (37), Dia1 in stress fiber formation (81), and WAVE2 in lamellipodium extension (72). Thus, Mena is a better candidate as a partner for IRSp53-mediated filopodia than Dia1 or WAVE2.There is good evidence for IRSp53 as a cellular partner for Eps8 (19). Eps8 is an adaptor protein containing an N-terminal PTB domain that can associate with receptor tyrosine kinases (65), and perhaps β integrins (13), and a C-terminal SH3 domain that can associate with Abi1 (30). Binding of the general adaptor Abi1 appears to positively regulate the actin-capping domain at the C terminus of Eps8 (18). It has been suggested that IRSp53 and Eps8 as a complex regulate cell motility, and perhaps Rac1 activation, via SOS (24); more recently, their roles in filopodium formation have been addressed (19). The involvement of IRSp53, but not MIM-B or IRTKS, in filopodium formation might be related to its role as a Cdc42 effector. We show here that, surprisingly, the CRIB motif is not essential for this activity, but rather, the ability of IRSp53 to associate via its SH3 domain is required, and that this domain is controlled by 14-3-3 binding.We have focused on the regulation of Cdc42 effectors that bind 14-3-3, including IRSp53 and PAK4, which are found as 14-3-3 targets in various proteomic projects (32, 44). In this study, we characterize the binding of 14-3-3 to IRSp53 and uncover how this activity regulates IRSp53 function. The phosphorylation-dependent 14-3-3 binding is GSK3β dependent, and 14-3-3 blocks the accessibility of both the CRIB and SH3 domains of IRSp53, thus indicating its primary function in controlling IRSp53 partners. This regulation of the SH3 domain by 14-3-3 is critical in the proper localization and termination of IRSp53 function to promote filopodium dynamics.     

Quantitative Fluorescence Resonance Energy Transfer Microscopy Analysis of the Human Immunodeficiency Virus Type 1 Gag-Gag Interaction: Relative Contributions of the CA and NC Domains and Membrane Binding

The human immunodeficiency virus type 1 structural polyprotein Pr55^Gag is necessary and sufficient for the assembly of virus-like particles on cellular membranes. Previous studies demonstrated the importance of the capsid C-terminal domain (CA-CTD), nucleocapsid (NC), and membrane association in Gag-Gag interactions, but the relationships between these factors remain unclear. In this study, we systematically altered the CA-CTD, NC, and the ability to bind membrane to determine the relative contributions of, and interplay between, these factors. To directly measure Gag-Gag interactions, we utilized chimeric Gag-fluorescent protein fusion constructs and a fluorescence resonance energy transfer (FRET) stoichiometry method. We found that the CA-CTD is essential for Gag-Gag interactions at the plasma membrane, as the disruption of the CA-CTD has severe impacts on FRET. Data from experiments in which wild-type (WT) and CA-CTD mutant Gag molecules are coexpressed support the idea that the CA-CTD dimerization interface consists of two reciprocal interactions. Mutations in NC have less-severe impacts on FRET between normally myristoylated Gag proteins than do CA-CTD mutations. Notably, when nonmyristoylated Gag interacts with WT Gag, NC is essential for FRET despite the presence of the CA-CTD. In contrast, constitutively enhanced membrane binding eliminates the need for NC to produce a WT level of FRET. These results from cell-based experiments suggest a model in which both membrane binding and NC-RNA interactions serve similar scaffolding functions so that one can functionally compensate for a defect in the other.The human immunodeficiency virus type 1 (HIV-1) structural precursor polyprotein Pr55^Gag is necessary and sufficient for the assembly of virus-like particles (VLPs). Gag is composed of four major structural domains, matrix (MA), capsid (CA), nucleocapsid (NC), and p6, as well as two spacer peptides, SP1 and SP2 (3, 30, 94). Following particle assembly and release, cleavage by HIV-1 protease separates these domains. However, these domains must work together in the context of the full-length Gag polyprotein to drive particle assembly.Previous studies have mapped two major functional domains involved in the early steps of assembly: first, Gag associates with cellular membranes via basic residues and N-terminal myristoylation of the MA domain (10, 17, 20, 35, 39, 87, 91, 106); second, the Gag-Gag interaction domains that span the CA C-terminal domain (CA-CTD) and NC domain promote Gag multimerization (3, 11, 14, 16, 18, 23, 27, 29, 30, 33, 36, 46, 64, 88, 94, 102, 103). Structural and genetic studies have identified two residues (W184 and M185) within a dimerization interface in the CA-CTD that are critical to CA-CA interactions (33, 51, 74, 96). Analytical ultracentrifugation of heterodimers formed between wild-type (WT) Gag and Gag mutants with changes at these residues suggests that the dimerization interface consists of two reciprocal interactions, one of which can be disrupted to form a “half-interface” (22).In addition to the CA-CTD, NC contributes to assembly via 15 basic residues (8, 9, 11, 14, 18, 23, 25, 28, 34, 40, 43, 54, 57, 58, 74, 79, 88, 97, 104, 105), although some researchers have suggested that NC instead contributes to the stability of mature virions after assembly (75, 98, 99). It is thought that the contribution of NC to assembly is due to its ability to bind RNA, since the addition of RNA promotes the formation of particles in vitro (14-16, 37, 46), and RNase treatment disrupts Gag-Gag interactions (11) and immature viral cores (67). However, RNA is not necessary per se, since dimerization motifs can substitute for NC (1, 4, 19, 49, 105). This suggests a model in which RNA serves a structural role, such as a scaffold, to promote Gag-Gag interactions through NC. Based on in vitro studies, it has been suggested that this RNA scaffolding interaction facilitates the low-order Gag multimerization mediated by CA-CTD dimerization (4, 37, 49, 62, 63, 85). Despite a wealth of biochemical data, the relative contributions of the CA-CTD and NC to Gag multimerization leading to assembly are yet to be determined in cells.Mutations in Gag interaction domains alter membrane binding in addition to affecting Gag multimerization. In particular, mutations or truncations of CA reduce membrane binding (21, 74, 82), and others previously reported that mutations or truncations of NC affect membrane binding (13, 78, 89, 107). These findings are consistent with a myristoyl switch model of membrane binding in which Gag can switch between high- and low-membrane-affinity states (38, 71, 76, 83, 86, 87, 92, 95, 107). Many have proposed, and some have provided direct evidence (95), that Gag multimerization mediated by CA or NC interactions promotes the exposure of the myristoyl moiety to facilitate membrane associations.Gag membrane binding and multimerization appear to be interrelated steps of virus assembly, since membrane binding also facilitates Gag multimerization. Unlike betaretroviruses that fully assemble prior to membrane targeting and envelopment (type B/D), lentiviruses, such as HIV, assemble only on cellular membranes at normal Gag expression levels (type C), although non-membrane-bound Gag complexes exist (45, 58, 60, 61, 65). Consistent with this finding, mutations that reduce Gag membrane associations cause a defect in Gag multimerization (59, 74). Therefore, in addition to their primary effects on Gag-Gag interactions, mutations in Gag interaction domains cause a defect in membrane binding, which, in turn, causes a secondary multimerization defect. To determine the relative contributions of the CA-CTD and the NC domain to Gag-Gag interactions at the plasma membrane, it is essential to eliminate secondary effects due to a modulation of membrane binding.Except for studies using a His-tag-mediated membrane binding system (5, 46), biochemical studies of C-type Gag multimerization typically lack membranes. Therefore, these studies do not fully represent particle assembly, which occurs on biological membranes in cells. Furthermore, many biochemical and structural approaches are limited to isolated domains or truncated Gag constructs. Thus, some of these studies are perhaps more relevant to the behavior of protease-cleaved Gag in mature virions. With few exceptions (47, 74), cell-based studies of Gag multimerization have typically been limited to measuring how well mutant Gag is incorporated into VLPs when coexpressed or not with WT Gag. Since VLP production is a complex multistep process, effects of mutations on other steps in the process can confound this indirect measure. For example, NC contributes to VLP production by both promoting multimerization and interacting with the host factor ALIX to promote VLP release (26, 80). To directly assay Gag multimerization in cells, several groups (24, 45, 52, 56) developed microscopy assays based on fluorescence resonance energy transfer (FRET). These assays measure the transfer of energy between donor and acceptor fluorescent molecules that are brought within ∼5 nm by the association of the proteins to which they are attached (41, 48, 90). However, these microscopy-based Gag FRET assays have not been used to fully elucidate several fundamental aspects of HIV-1 Gag multimerization at the plasma membrane of cells, such as the relative contributions of the CA-CTD and NC and the effect of membrane binding on Gag-Gag interactions. In this study, we used a FRET stoichiometry method based on calibrated spectral analysis of fluorescence microscopy images (41). This algorithm determines the fractions of both donor and acceptor fluorescent protein-tagged Gag molecules participating in FRET. For cells expressing Gag molecules tagged with donor (cyan fluorescent protein [CFP]) and acceptor (yellow fluorescent protein [YFP]) molecules, this method measures the apparent FRET efficiency, which is proportional to the mole fraction of Gag constructs in complex. By measuring apparent FRET efficiencies, quantitative estimates of the mole fractions of interacting proteins can be obtained.Using this FRET-based assay, we aim to answer two questions: (i) what are the relative contributions of CA-CTD and NC domains to Gag multimerization when secondary effects via membrane binding are held constant, and (ii) what is the effect of modulating membrane binding on the ability of Gag mutants to interact with WT Gag?Our data demonstrate that the CA-CTD dimerization interface is essential for Gag multimerization at the plasma membrane, as fully disrupting the CA-CTD interaction abolishes FRET, whereas a modest level of FRET is still detected in the absence of NC. We also present evidence that the CA-CTD dimerization interface consists of two reciprocal interactions, allowing the formation of a half-interface that can still contribute to Gag multimerization. Notably, when Gag derivatives with an intact CA-CTD were coexpressed with WT Gag, either membrane binding ability or NC was required for the Gag mutants to interact with WT Gag, suggesting functional compensation between these factors.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

The Amino Terminus of Herpes Simplex Virus Type 1 Glycoprotein K (gK) Modulates gB-Mediated Virus-Induced Cell Fusion and Virion Egress

Herpes simplex virus type 1 (HSV-1)-induced cell fusion is mediated by viral glycoproteins and other membrane proteins expressed on infected cell surfaces. Certain mutations in the carboxyl terminus of HSV-1 glycoprotein B (gB) and in the amino terminus of gK cause extensive virus-induced cell fusion. Although gB is known to be a fusogenic glycoprotein, the mechanism by which gK is involved in virus-induced cell fusion remains elusive. To delineate the amino-terminal domains of gK involved in virus-induced cell fusion, the recombinant viruses gKΔ31-47, gKΔ31-68, and gKΔ31-117, expressing gK carrying in-frame deletions spanning the amino terminus of gK immediately after the gK signal sequence (amino acids [aa] 1 to 30), were constructed. Mutant viruses gKΔ31-47 and gKΔ31-117 exhibited a gK-null (ΔgK) phenotype characterized by the formation of very small viral plaques and up to a 2-log reduction in the production of infectious virus in comparison to that for the parental HSV-1(F) wild-type virus. The gKΔ31-68 mutant virus formed substantially larger plaques and produced 1-log-higher titers than the gKΔ31-47 and gKΔ31-117 mutant virions at low multiplicities of infection. Deletion of 28 aa from the carboxyl terminus of gB (gBΔ28syn) caused extensive virus-induced cell fusion. However, the gBΔ28syn mutation was unable to cause virus-induced cell fusion in the presence of the gKΔ31-68 mutation. Transient expression of a peptide composed of the amino-terminal 82 aa of gK (gKa) produced a glycosylated peptide that was efficiently expressed on cell surfaces only after infection with the HSV-1(F), gKΔ31-68, ΔgK, or UL20-null virus. The gKa peptide complemented the gKΔ31-47 and gKΔ31-68 mutant viruses for infectious-virus production and for gKΔ31-68/gBΔ28syn-mediated cell fusion. These data show that the amino terminus of gK modulates gB-mediated virus-induced cell fusion and virion egress.Herpes simplex virus type 1 (HSV-1) specifies at least 11 virally encoded glycoproteins, as well as several nonglycosylated and lipid-anchored membrane-associated proteins, which serve important functions in virion infectivity and virus spread. Although cell-free enveloped virions can efficiently spread viral infection, virions can also spread by causing cell fusion of adjacent cellular membranes. Virus-induced cell fusion, which is caused by viral glycoproteins expressed on infected cell surfaces, enables transmission of virions from one cell to another, avoiding extracellular spaces and exposure of free virions to neutralizing antibodies (reviewed in reference 56). Most mutations that cause extensive virus-induced cell-to-cell fusion (syncytial or syn mutations) have been mapped to at least four regions of the viral genome: the UL20 gene (5, 42, 44); the UL24 gene (37, 58); the UL27 gene, encoding glycoprotein B (gB) (9, 51); and the UL53 gene, coding for gK (7, 15, 35, 53, 54, 57).Increasing evidence suggests that virus-induced cell fusion is mediated by the concerted action of glycoproteins gD, gB, and gH/gL. Recent studies have shown that gD interacts with both gB and gH/gL (1, 2). Binding of gD to its cognate receptors, including Nectin-1, HVEM, and others (12, 29, 48, 59, 60, 62, 63), is thought to trigger conformation changes in gH/gL and gB that cause fusion of the viral envelope with cellular membranes during virus entry and virus-induced cell fusion (32, 34). Transient coexpression of gB, gD, and gH/gL causes cell-to-cell fusion (49, 68). However, this phenomenon does not accurately model viral fusion, because other viral glycoproteins and membrane proteins known to be important for virus-induced cell fusion are not required (6, 14, 31). Specifically, gK and UL20 were shown to be absolutely required for virus-induced cell fusion (21, 46). Moreover, syncytial mutations within gK (7, 15, 35, 53, 54, 57) or UL20 (5, 42, 44) promote extensive virus-induced cell fusion, and viruses lacking gK enter more slowly than wild-type virus into susceptible cells (25). Furthermore, transient coexpression of gK carrying a syncytial mutation with gB, gD, and gH/gL did not enhance cell fusion, while coexpression of the wild-type gK with gB, gD, and gH/gL inhibited cell fusion (3).Glycoproteins gB and gH are highly conserved across all subfamilies of herpesviruses. gB forms a homotrimeric type I integral membrane protein, which is N glycosylated at multiple sites within the polypeptide. An unusual feature of gB is that syncytial mutations that enhance virus-induced cell fusion are located exclusively in the carboxyl terminus of gB, which is predicted to be located intracellularly (51). Single-amino-acid substitutions within two regions of the intracellular cytoplasmic domain of gB were shown to cause syncytium formation and were designated region I (amino acid [aa] positions 816 and 817) and region II (aa positions 853, 854, and 857) (9, 10, 28, 69). Furthermore, deletion of 28 aa from the carboxyl terminus of gB, disrupting the small predicted alpha-helical domain H17b, causes extensive virus-induced cell fusion as well as extensive glycoprotein-mediated cell fusion in the gB, gD, and gH/gL transient-coexpression system (22, 49, 68). The X-ray structure of the ectodomain of gB has been determined and is predicted to assume at least two major conformations, one of which may be necessary for the fusogenic properties of gB. Therefore, perturbation of the carboxyl terminus of gB may alter the conformation of the amino terminus of gB, thus favoring one of the two predicted conformational structures that causes membrane fusion (34).The UL53 (gK) and UL20 genes encode multipass transmembrane proteins of 338 and 222 aa, respectively, which are conserved in all alphaherpesviruses (15, 42, 55). Both proteins have multiple sites where posttranslational modification can occur; however, only gK is posttranslationally modified by N-linked carbohydrate addition (15, 35, 55). The specific membrane topologies of both gK and UL20 protein (UL20p) have been predicted and experimentally confirmed using epitope tags inserted within predicted intracellular and extracellular domains (18, 21, 44). Syncytial mutations in gK map predominantly within extracellular domains of gK and particularly within the amino-terminal portion of gK (domain I) (18), while syncytial mutations of UL20 are located within the amino terminus of UL20p, shown to be located intracellularly (44). A series of recent studies have shown that HSV-1 gK and UL20 functionally and physically interact and that these interactions are necessary for their coordinate intracellular transport and cell surface expression (16, 18, 21, 26, 45). Specifically, direct protein-protein interactions between the amino terminus of HSV-1 UL20 and gK domain III, both of which are localized intracellularly, were recently demonstrated by two-way coimmunoprecipitation experiments (19).According to the most prevalent model for herpesvirus intracellular morphogenesis, capsids initially assemble within the nuclei and acquire a primary envelope by budding into the perinuclear spaces. Subsequently, these virions lose their envelope through fusion with the outer nuclear lamellae. Within the cytoplasm, tegument proteins associate with the viral nucleocapsid and final envelopment occurs by budding of cytoplasmic capsids into specific trans-Golgi network (TGN)-associated membranes (8, 30, 47, 70). Mature virions traffic to cell surfaces, presumably following the cellular secretory pathway (33, 47, 61). In addition to their significant roles in virus-induced cell fusion, gK and UL20 are required for cytoplasmic virion envelopment. Viruses with deletions in either the gK or the UL20 gene are unable to translocate from the cytoplasm to extracellular spaces and accumulated as unenveloped virions in the cytoplasm (5, 15, 20, 21, 26, 35, 36, 38, 44, 55). Current evidence suggests that the functions of gK and UL20 in cytoplasmic virion envelopment and virus-induced cell fusion are carried out by different, genetically separable domains of UL20p. Specifically, UL20 mutations within the amino and carboxyl termini of UL20p allowed cotransport of gK and UL20p to cell surfaces, virus-induced cell fusion, and TGN localization, while effectively inhibiting cytoplasmic virion envelopment (44, 45).In this paper, we demonstrate that the amino terminus of gK expressed as a free peptide of 82 aa (gKa) is transported to infected cell surfaces by viral proteins other than gK or UL20p and facilitates virus-induced cell fusion caused by syncytial mutations in the carboxyl terminus of gB. Thus, functional domains of gK can be genetically separated, as we have shown previously (44, 45), as well as physically separated into different peptide portions that retain functional activities of gK. These results are consistent with the hypothesis that the amino terminus of gK directly or indirectly interacts with and modulates the fusogenic properties of gB.