Serine Dephosphorylation of Receptor Protein Tyrosine Phosphatase α in Mitosis Induces Src Binding and Activation

Receptor protein tyrosine phosphatase α (RPTPα) is the mitotic activator of the protein tyrosine kinase Src. RPTPα serine hyperphosphorylation was proposed to mediate mitotic activation of Src. We raised phosphospecific antibodies to the two main serine phosphorylation sites, and we discovered that RPTPα Ser204 was almost completely dephosphorylated in mitotic NIH 3T3 and HeLa cells, whereas Ser180 and Tyr789 phosphorylation were only marginally reduced in mitosis. Concomitantly, Src pTyr527 and pTyr416 were dephosphorylated, resulting in 2.3-fold activation of Src in mitosis. Using inhibitors and knockdown experiments, we demonstrated that dephosphorylation of RPTPα pSer204 in mitosis was mediated by PP2A. Mutation of Ser204 to Ala did not activate RPTPα, and intrinsic catalytic activity of RPTPα was not affected in mitosis. Interestingly, binding of endogenous Src to RPTPα was induced in mitosis. GRB2 binding to RPTPα, which was proposed to compete with Src binding to RPTPα, was only modestly reduced in mitosis, which could not account for enhanced Src binding. Moreover, we demonstrate that Src bound to mutant RPTPα-Y789F, lacking the GRB2 binding site, and mutant Src with an impaired Src homology 2 (SH2) domain bound to RPTPα, illustrating that Src binding to RPTPα is not mediated by a pTyr-SH2 interaction. Mutation of RPTPα Ser204 to Asp, mimicking phosphorylation, reduced coimmunoprecipitation with Src, suggesting that phosphorylation of Ser204 prohibits binding to Src. Based on our results, we propose a new model for mitotic activation of Src in which PP2A-mediated dephosphorylation of RPTPα pSer204 facilitates Src binding, leading to RPTPα-mediated dephosphorylation of Src pTyr527 and pTyr416 and hence modest activation of Src.Protein tyrosine phosphatases (PTPs) are responsible for dephosphorylation of the phosphotyrosyl residues. The human genome contains approximately 100 genes that encode members of the four PTP families, and most of them have mouse orthologues (2, 48). According to their subcellular localization, the classical PTPs, encoded by less than half of the total PTP genes, are divided into two subfamilies: cytoplasmic and receptor protein tyrosine phosphatases (RPTPs). The majority of the RPTPs contain, besides a variable extracellular domain and a transmembrane domain, two highly homologous phosphatase domains (27), with the membrane-proximal domain comprising most of the catalytic activity (33).RPTPα is a typical RPTP with a small, highly glycosylated extracellular domain (13). RPTPα function is regulated by many mechanisms, including proteolysis (18), oxidation (55), dimerization (7, 23, 24, 47, 52), and phosphorylation of serine and tyrosine residues (16, 17, 49). RPTPα is broadly expressed in many cell types, and over the years, RPTPα has been shown to be involved in a number of signaling mechanisms, including neuronal (15) and skeletal muscle (34) cell differentiation, neurite elongation (8, 9, 56), insulin receptor signaling downregulation (3, 28, 30, 31, 35), insulin secretion (25), activation of voltage-gated potassium channel Kv1.2 (51), long-term potentiation in hippocampal neurons (32, 38), matrix-dependent force transduction (53), and cell spreading and migration (21, 45, 57).The majority of the roles played in these cellular processes involve RPTPα''s ability to activate the proto-oncogenes Src and Fyn by dephosphorylating their C-terminal inhibitory phosphotyrosine (5, 15, 39, 45, 61). Normally, this phosphotyrosine (pTyr527 in chicken Src) binds to the Src homology 2 (SH2) domain, keeping the protein in an inactive closed conformation. A displacement mechanism was proposed for RPTPα-mediated Src activation in which pTyr789 of RPTPα is required to bind the SH2 domain of Src before RPTPα dephosphorylates Tyr527 (58). This model is the subject of debate since other studies show that RPTPα lacking Tyr789 is still able to dephosphorylate and activate Src (12, 26, 29, 56). In normal cells, Src reaches its activation peak during mitosis (4, 11, 40, 42), and with the help of overexpressing cells, it was shown that this activation is triggered mainly by RPTPα. The model that emerged is that RPTPα is activated in mitosis due to serine hyperphosphorylation and detaches from the GRB2 scaffolding protein (59, 60) that normally binds most of the pTyr789 of RPTPα via its SH2 domain (14, 17, 46). Two serine phosphorylation sites were mapped in the juxtamembrane domain of RPTPα, Ser180 and Ser204 (49). The kinases that were found responsible for their phosphorylation were protein kinase C delta (PKCdelta) (10) and CaMKIIalpha (9), but there is no clear evidence that these kinases are activated in mitosis. We set out to investigate the role of serine phosphorylation of RPTPα in mitotic activation of Src.We generated phosphospecific antibodies and show that RPTPα pSer204, but not pSer180, is dephosphorylated in mitotic NIH 3T3 and HeLa cells, concomitantly with activation of Src. Selective inhibitors suggested that PP2A was the phosphatase that dephosphorylated pSer204. RNA interference (RNAi)-mediated knockdown of the catalytic subunit of PP2A demonstrated that indeed PP2A was responsible for mitotic dephosphorylation of RPTPα pSer204. It is noteworthy that PP2A is known to be activated in mitosis. Intrinsic PTP activities of RPTPα were similar in unsynchronized and mitotic cells, and mutation of Ser204 did not activate RPTPα in in vitro PTP assays. Yet, Src binding to RPTPα was induced in mitotic NIH 3T3 cells and RPTPα-S204D with a phosphomimicking mutation at Ser204 coimmunoprecipitated less efficiently with Src. Based on our results, we propose a mechanism for mitotic activation of Src that is triggered by dephosphorylation of RPTPα pSer204, resulting in enhanced affinity for Src and subsequent dephosphorylation and activation of Src.     

Dynamin Reduces Pyk2 Y402 Phosphorylation and Src Binding in Osteoclasts

Signaling via the Pyk2-Src-Cbl complex downstream of integrins contributes to the assembly, organization, and dynamics of podosomes, which are the transient adhesion complexes of highly motile cells such as osteoclasts and dendritic cells. We previously demonstrated that the GTPase dynamin is associated with podosomes, regulates actin flux in podosomes, and promotes bone resorption by osteoclasts. We report here that dynamin associates with Pyk2, independent of dynamin''s GTPase activity, and reduces Pyk2 Y402 phosphorylation in a GTPase-dependent manner, leading to decreased Src binding to Pyk2. Overexpressing dynamin decreased the macrophage colony-stimulating factor- and adhesion-induced phosphorylation of Pyk2 in osteoclastlike cells, suggesting that dynamin is likely to regulate Src-Pyk2 binding downstream of integrins and growth factor receptors with important cellular consequences. Furthermore, catalytically active Src promotes dynamin-Pyk2 association, and mutating specific Src-phosphorylated tyrosine residues in dynamin blunts the dynamin-induced decrease in Pyk2 phosphorylation. Thus, since Src binds to Pyk2 through its interaction with phospho-Y402, our results suggest that Src activates a negative-feedback loop downstream of integrin engagement and other stimuli by promoting both the binding of dynamin to Pyk2-containing complexes and the dynamin-dependent decrease in Pyk2 Y402 phosphorylation, ultimately leading to the dissociation of Src from Pyk2.Podosomes are specialized transient actin-containing adhesion structures (11, 14, 37, 60) that are found in highly motile cells, such as osteoclasts, macrophages, dendritic cells, transformed metastatic cells, and v-src-transformed cells (37, 43), where they are thought to play important roles in cellular migration and invasion (34). In resorbing osteoclasts on bone, podosomes are concentrated within the sealing zone, a beltlike actin-rich structure that is important for adhesion and which delineates the resorptive region of the cell known as the ruffled border. Unlike focal adhesions, which are relatively stable structures (11, 60), the assembly and disassembly of podosomes occurs within minutes (t_1/2 = 2 to 4 min) and involves the recruitment and activation of integrins, signaling proteins and scaffolding proteins (11, 14, 35, 47, 60). However, the mechanisms of action of key signaling proteins involved in podosome assembly and disassembly are only partially understood.The focal adhesion kinase Pyk2 has been linked to the proliferation, migration, and activity of a variety of mesenchymal, epithelial, and hematopoietic cell types. Several groups, including our own, have reported the importance of Pyk2 in podosome belt organization, cell spreading, and bone-resorbing activity in osteoclasts (18, 26, 31, 40, 65, 66). Pyk2 is recruited to activated β₂ and β₃ integrins (9, 20) at adhesion sites and is autophosphorylated at Y402 (17, 47, 50) via an intermolecular trans-acting mechanism (46). Although Pyk2 is partially activated by integrin-induced Ca²⁺ signaling (20, 50), the induction of Pyk2''s full catalytic activity requires the binding of Src via its SH2 domain to autophosphorylated Pyk2 Y402 and the subsequent phosphorylation of Pyk2 at functionally distinct sites, including Y579, Y580, and Y881 (17, 31, 46). The binding of Src to phosphorylated Pyk2, which leads to the formation of a multiprotein signaling complex at adhesion sites (17, 40, 50), is critical for Pyk2 activity, as demonstrated by the fact that Pyk2 phosphorylation and activity are significantly reduced in osteoclasts derived from Src^−/− mice (17, 40). Src^−/− osteoclasts also exhibit decreased motility (50) and decreased bone-resorbing activity (40, 54, 59), and we recently demonstrated that Src promotes both podosome formation and disassembly, as well as actin flux into existing podosomes and the organization of podosomes into a peripheral belt in osteoclasts (15).We have also demonstrated that the GTP-hydrolyzing protein dynamin-2, which is ubiquitously expressed and well known for its role in endocytosis (53), regulates actin remodeling in the podosomes of osteoclasts and Rous sarcoma virus-transformed baby hamster kidney cells (43). In addition, a dynamin-2 mutant that binds GTP with reduced affinity (dynK44A) (12) decreased the flux of actin into podosomes (43) and disrupted podosome belt formation in osteoclasts, thereby affecting osteoclast migration and bone-resorbing activity (8). The dynamin proteins, of which there are three homologous isoforms (3), contain several protein domains: a GTP-hydrolyzing domain (GTPase), a plextrin homology domain that mediates binding to phosphoinositides, a GTPase effector domain (GED), and a C-terminal proline-rich domain (PRD) (38, 45, 55) through which dynamin binds a number of functionally diverse SH3-containing molecules, such as Src, cortactin, Grb2, and N-Wasp (1, 7, 27, 39, 58). We previously reported that dynamin-2 partially colocalizes and associates with the E3-ubiquitin ligase Cbl within the podosome belt/sealing zone of osteoclasts, as well as in SYF cells, which lack the Src family kinases Src, Yes, and Fyn, and in HEK 293 cells that stably express the vitronectin receptor (293VnR) (8). Protein complexes containing dynamin-2 and Cbl, which are both substrates of Src (1, 2, 23, 50, 56), were disrupted in the presence of activated Src and stabilized in the absence of Src (8), demonstrating a key role of Src in regulating the formation of signaling complexes in osteoclasts downstream of integrins.In the present study, we sought to determine whether dynamin, which regulates podosome actin dynamics and bone resorption in osteoclasts, also associates with Pyk2 and/or regulates Pyk2''s activities in osteoclasts. We report here that dynamin associates with Pyk2 and promotes the dephosphorylation of Pyk2 Y402 and that catalytically active Src promotes both dynamin''s association with Pyk2 and the dynamin-induced dephosphorylation of Pyk2 Y402, resulting, in turn, in the decreased binding of Src to Pyk2. Thus, we propose that dynamin regulates podosome dynamics and osteoclast bone-resorbing activity by promoting the disassembly of the Pyk2-Src-Cbl complex that is formed in osteoclasts downstream of β₃ integrin activation.     

Myristoylation and Membrane Binding Regulate c-Src Stability and Kinase Activity

Myristoylation is critical for membrane association of Src kinases, but a role for myristate in regulating other aspects of Src biology has not been explored. In the c-Abl tyrosine kinase, myristate binds within a hydrophobic pocket at the base of the kinase domain and latches the protein into an autoinhibitory conformation. A similar pocket has been predicted to exist in c-Src, raising the possibility that Src might also be regulated by myristoylation. Here we show that in contrast to the case for c-Abl, myristoylation exerts a positive effect on c-Src kinase activity. We also demonstrate that myristoylation and membrane binding regulate c-Src ubiquitination and degradation. Nonmyristoylated c-Src exhibited reduced kinase activity but had enhanced stability compared to myristoylated c-Src. We then mutated critical residues in the predicted myristate binding pocket of c-Src. Mutation of L360 and/or E486 had no effect on c-Src membrane binding or localization. However, constructs containing a T456A mutation were partially released from the membrane, suggesting that mutagenesis could induce c-Src to undergo an artificial myristoyl switch. All of the pocket mutants exhibited decreased kinase activity. We concluded that myristoylation and the pocket residues regulate c-Src, but in a manner very different from that for c-Abl.Src family kinases (SFKs) are nonreceptor tyrosine kinases that act as key mediators of cellular signal transduction (12). The nine SFK members, Src, Yes, Fyn, Hck, Lck, Lyn, Blk, Fgr, and Yrk, play crucial roles in cellular proliferation, survival, migration, and growth factor and cytokine stimulation pathways (26, 39, 56). All SFKs share a similar domain arrangement, consisting of SH3, SH2, and kinase (SH1) domains as well as a unique domain and a membrane-targeting SH4 region at the N terminus (11). Crystal structures have shown that the catalytic activity of SFKs is tightly regulated by autoinhibition. The SH3 domain binds to a polyproline region in the linker between the SH2 and kinase domains, and the SH2 domain binds to a phosphotyrosine residue (Tyr527 in avian c-Src) near the C terminus. Kinase activation can be achieved by displacing one or all of the autoinhibitory interactions (52, 59, 60).All SFKs are myristoylated at the N terminus (47). Myristoylation occurs cotranslationally and is catalyzed by the enzyme N-myristoyl transferase (NMT) (19). The 14-carbon saturated fatty acid myristate is covalently attached to the N-terminal glycine residue via an amide bond, making myristoylation an essentially irreversible modification (44, 48, 49). Myristoylation is necessary but not sufficient to anchor a protein to the membrane, and membrane binding of myristoylated proteins requires a second signal. For Src, the second signal is a polybasic cluster of amino acids that interacts with acidic phospholipids on the inner leaflet of the membrane bilayer (34, 35, 44, 46, 53). Nearly all other SFKs are instead modified by attachment of the 16-carbon saturated fatty acid palmitate to cysteine residues 3 and 5 or 6 at the N terminus (48). Myristoylation and palmitoylation together form a “dual signal” motif that targets SFKs to membranes.Membrane binding is crucial for cellular functions mediated by Src and other SFKs. Nonmyristoylated forms of Src are cytoplasmic and cannot induce cellular transformation (14, 28, 29, 33). Membrane localization of c-Src has been shown to be important for dephosphorylation of Tyr527 and for mitotic activation of c-Src kinase activity (8), presumably because the phosphatase that acts on Tyr527 is membrane bound. Myristoylation has also been proposed to play a role in regulating nuclear transport of c-Src (17).For some myristoylated proteins, the myristate moiety can exist in two different conformational states, either sequestered inside a hydrophobic pocket within the protein or exposed and available for membrane binding (44, 48). Binding to a ligand or another protein can cause a switch from one state to another, resulting in membrane association or dissociation. “Myristoyl switch” mechanisms have been identified in a variety of myristoylated proteins, including recoverin and HIV-1 Gag (4-6, 43, 45, 55). In the c-Abl tyrosine kinase, a “myristoyl phosphotyrosine” switch is operative. Myristate binds within a hydrophobic pocket at the base of the c-Abl kinase domain, docking the SH2 domain against the kinase domain in such a way that it prevents activation of the kinase by phosphotyrosine ligands (22, 36). A similar pocket is predicted to exist at the base of the c-Src kinase domain, raising the possibility that c-Src in the autoinhibited form might be capable of binding its own N-terminal myristate group in a manner similar to that of c-Abl (16). To date, only nonmyristoylated, N-terminally truncated forms of c-Src have been crystallized, and the position of the myristate within the full-length c-Src protein is not known. A recent study provided support for the existence of a potential myristate binding pocket within c-Src (16). Exogenous addition of myristate to the Tyr527-phosphorylated form of nonmyristoylated c-Src induced chemical shift changes in the nuclear magnetic resonance (NMR) spectra for both the protein and the fatty acid. However, the site of myristate binding was not determined. Thus, it is still not known how or if myristate regulates c-Src kinase activity and whether the predicted myristate binding pocket functions within c-Src in a manner similar to that of c-Abl.In this study, we directly analyzed the role of myristoylation in regulating c-Src kinase activity and tested whether residues in the predicted myristate binding pocket contribute to myristate binding and/or c-Src activity. Here we show that myristoylation plays a positive role in regulating c-Src kinase activity. In contrast to c-Abl, nonmyristoylated c-Src exhibits reduced kinase activity both in vitro and in cells. We also made the surprising finding that the myristoylation status of c-Src determines its intracellular stability by regulating c-Src ubiquitination and degradation of the E3 ligase Cbl. Lastly, we analyzed the role of the predicted myristate binding pocket at the base of the c-Src kinase domain. Mutations in the pocket region resulted in decreased kinase activity and, with the exception of one mutation (Thr456Ala), had no effect on membrane binding of c-Src. We concluded that c-Src kinase activity is regulated by myristoylation, but in a different manner from that of c-Abl, and that a “myristoyl switch” is unlikely to be operative within c-Src.     

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.     

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.     

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.     

Contributions of F-BAR and SH2 Domains of Fes Protein Tyrosine Kinase for Coupling to the Fc?RI Pathway in Mast Cells

This study investigates the roles of Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) and SH2 domains of Fes protein tyrosine kinase in regulating its activation and signaling downstream of the high-affinity immunoglobulin G (IgE) receptor (FcɛRI) in mast cells. Homology modeling of the Fes F-BAR domain revealed conservation of some basic residues implicated in phosphoinositide binding (R113/K114). The Fes F-BAR can bind phosphoinositides and induce tubulation of liposomes in vitro. Mutation of R113/K114 to uncharged residues (RK/QQ) caused a significant reduction in phosphoinositide binding in vitro and a more diffuse cytoplasmic localization in transfected COS-7 cells. RBL-2H3 mast cells expressing full-length Fes carrying the RK/QQ mutation show defects in FcɛRI-induced Fes tyrosine phosphorylation and degranulation compared to cells expressing wild-type Fes. This correlated with reduced localization to Lyn kinase-containing membrane fractions for the RK/QQ mutant compared to wild-type Fes in mast cells. The Fes SH2 domain also contributes to Fes signaling in mast cells, via interactions with the phosphorylated FcɛRI β chain and the actin regulatory protein HS1. We show that Fes phosphorylates C-terminal tyrosine residues in HS1 implicated in actin stabilization. Thus, coordinated actions of the F-BAR and SH2 domains of Fes allow for coupling to FcɛRI signaling and potential regulation the actin reorganization in mast cells.Mast cells reside in connective and mucosal tissues and play a key protective role in the immune response to helminth infection (13, 21), sepsis (39), and snake or bee venoms (42). Mast cells express FcɛRI, which becomes sensitized to antigens or allergens upon immunoglobulin E (IgE) binding. Aggregation of FcɛRI by multivalent antigens causes the release of preformed mediators by degranulation and the de novo production of lipid mediators and cytokines (1, 52). Release of these mediators causes increased vascular permeability, leukocyte recruitment and activation, and inflammation (41). Aberrant mast cell activation is implicated in IgE-mediated type I hypersensitivity reactions including anaphylaxis, allergic rhinitis, and asthma (20). FcɛRI is a tetrameric receptor composed of an IgE-binding α chain and of β and γ chains containing immunoreceptor tyrosine-based activation motifs that become phosphorylated following multivalent antigen-mediated clustering of FcɛRI and activation of Src family protein tyrosine kinases (PTKs), primarily involving Lyn (51). Lyn phosphorylates and activates both positive effectors of FcɛRI signaling (e.g., Syk PTK) and key negative regulators (e.g., Shp-1 and SHIP) that serve to limit mast cell activation (28, 46, 69).Fes (the mammalian orthologue of the v-Fps and v-Fes oncoproteins from avian [57, 58] and feline [15, 56] retroviruses) and Fer are closely related PTKs that become activated following FcɛRI aggregation in mast cells (10). Surprisingly, FcɛRI-induced tyrosine phosphorylation of Fes and Fer does not require their kinase activities (55) and is almost entirely dependent on Lyn (67). Through the use of transgenic mouse models, evidence for both unique and redundant functions for Fes and Fer has been described in regulating hematopoiesis (55) and limiting the innate immune response (22, 40, 50, 72). In mast cells, we have shown that Fer promotes activation of p38 mitogen-activated protein kinase and chemotaxis of mast cells (10). We also found that Fer and Fes PTKs contribute to FcɛRI-evoked phosphorylation of platelet-endothelial cell adhesion molecule 1 (PECAM-1) (67).Each of the Fes and Fer PTKs is composed of an N-terminal regulatory domain containing a Fer-CIP4 homology (FCH) domain followed by several predicted coiled-coils (CC), a central SH2 domain, and C-terminal PTK domain (19). It is worth noting that early studies pointed toward an important role for the N-terminal domain of v-Fps for its transforming activity and membrane localization (5, 63). Several recent studies have defined the FCH and first CC domain (amino acids 1 to 300) in Fer, CIP4, and other pombe Cdc15 homology (PCH) family adaptor proteins as an F-BAR domain (also termed extended FCH or EFC domain) (reviewed in references 3 and 9). The F-BAR domain was found to constitute a novel phosphoinositide-binding domain that can promote tubulation of liposomes in vitro and membranes in vivo (27, 33, 66). The crystal structures of F-BAR domains from several PCH adaptors were recently solved (27, 59). The F-BAR module was shown to consist of a triple helical bundle that forms a homodimer, with a concave surface rich in basic residues that have recently been shown to contact phospholipids in curved membranes (16). In vitro studies using the Fer F-BAR domain have shown that the F-BAR domain binds strongly to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂]; however, the Fer F-BAR is relatively weak compared with the adaptor protein FBP17 at inducing membrane tubulation (66). Liposome sedimentation assays have identified several conserved basic residues required for F-BAR domain binding to PI(4,5)P₂(66). The substitution of R113/K114 to glutamines (RK/QQ) in FBP17 reduced phosphoinositide binding by 80% (66). A recent electron cryoelectron microscopy study provided insights into binding of F-BAR dimers to flat and curved membranes via different binding faces (16). This study also confirmed that R113/K114 residues (in CIP4) constitute a site of direct interaction with the liposomes. Interestingly, microdomains of the plasma membrane rich in PI(4,5)P₂ are sites of dynamic actin assembly (47) and endocytosis (4, 31). Previous studies have described Fes localization to a variety of subcellular structures, including endocytic vesicles (71), the trans-Golgi apparatus (71), microtubules (37), and focal adhesions (44). The rapid activation of Fes and Fer PTKs upon FcɛRI aggregation on mast cells (10) would suggest that there is a mechanism by which Fes localizes at or near the plasma membrane. Phosphoinositide-binding via the F-BAR domain of Fes and Fer PTKs may promote their recruitment to the plasma membrane prior to their activation by cell surface receptors such as FcɛRI. The potential colocalization with endocytosis and actin assembly regulators may allow for regulation of receptor endocytosis or chemotaxis of mast cells by Fes/Fer PTKs. A recent study implicates Rab5 GTPase and its exchange factor RabGEF1/Rabex-5 in promoting internalization of FcɛRI following clustering by antigens (34). It is worth noting that defects in internalization of Toll-like receptor 4 and transferrin receptor were observed in Fes-deficient macrophages (48), and there is a potential role for Fes in regulating internalization of mast cell receptors.In this study, we provide novel insights into the phospholipid binding and liposome tubulating properties of the Fes F-BAR domain. Mutation of two conserved basic residues within the Fes F-BAR domain (RK/QQ) reduced phospholipid binding in vitro, and membrane localization in vivo. In transfected RBL-2H3 mast cells, the Fes harboring the RK/QQ mutation (Fes^RK/QQ) displayed reduced FcɛRI-evoked tyrosine phosphorylation compared to wild-type Fes (Fes^WT), which correlated with reduced localization to Lyn-containing membranes in mast cells. The SH2 domain of Fes was found to interact with several phosphoproteins in mast cells, including FcɛRI and HS1, an actin regulator and cortactin homologue. We found that Fes contributes to HS1 phosphorylation at C-terminal residues implicated in actin branch stabilization, and we present a model for how F-BAR-containing adaptor proteins and PTKs may coordinate actin-driven endocytosis in mast cells.     

DivIC Stabilizes FtsL against RasP Cleavage

The essential cell division protein FtsL is a substrate of the intramembrane protease RasP. Using heterologous coexpression experiments, we show here that the division protein DivIC stabilizes FtsL against RasP cleavage. Degradation seems to be initiated upon accessibility of a cytosolic substrate recognition motif.Cell division in bacteria is a highly regulated process (1). The division site selection as well as assembly and disassembly of the divisome have to be strictly controlled (1, 4). Although the spatial control of the divisome is relatively well understood (2, 4, 14, 17), mechanisms governing the temporal control of division are still mainly elusive. Regulatory proteolysis was thought to be a potential modulatory mechanism (8, 9). The highly unstable division protein FtsL was shown to be rate limiting for division and would make an ideal candidate for a regulatory factor in the timing of bacterial cell division (7, 9). In Bacillus subtilis, FtsL is an essential protein of the membrane part of the divisome (5, 7, 8). It is necessary for the assembly of the membrane-spanning division proteins, and a knockout is lethal (8, 9, 12). We have previously reported that FtsL is a substrate of the intramembrane protease RasP (5).These findings raised the question of whether RasP can regulate cell division by cleaving FtsL from the division complex. In order to mimic the situation in which FtsL is bound to at least one of its interaction partners, we used a heterologous coexpression system in which we synthesized FtsL and DivIC. It has been reported before that DivIC and FtsL are intimate binding partners in various organisms (6, 9, 15, 21, 22, 26) and that FtsL and DivIC (together with DivIB) can form complexes even in the absence of the other divisome components (6, 21). We therefore asked whether RasP is able to cleave FtsL in the presence of its major interaction partner DivIC, which would argue for the possibility that RasP could cleave FtsL within a mature divisome. In contrast, if interaction with DivIC could stabilize FtsL against RasP cleavage, this result would bring such a model into question. An alternative option for the role of RasP might be the removal of FtsL from the membrane. It has been shown that divisome disassembly and prevention of reassembly are crucial to prevent minicell formation close to the new cell poles (3, 16).     

Semaphorin 4D Signaling Requires the Recruitment of Phospholipase Cγ into the Plexin-B1 Receptor Complex

The semaphorin 4D (Sema4D) receptor plexin-B1 constitutively interacts with particular Rho guanine nucleotide exchange factors (RhoGEFs) and thereby mediates Sema4D-induced RhoA activation, a process which involves the tyrosine phosphorylation of plexin-B1 by ErbB-2. It is, however, unknown how plexin-B1 phosphorylation regulates RhoGEF activity. We show here that activation of plexin-B1 by Sema4D and its subsequent tyrosine phosphorylation creates docking sites for the SH2 domains of phospholipase Cγ (PLCγ). PLCγ is thereby recruited into the plexin-B1 receptor complex and via its SH3 domain activates the Rho guanine nucleotide exchange factor PDZ-RhoGEF. PLCγ-dependent RhoGEF activation is independent of its lipase activity. The recruitment of PLCγ has no effect on the R-Ras GTPase-activating protein activity of plexin-B1 but is required for Sema4D-induced axonal growth cone collapse as well as for the promigratory effects of Sema4D on cancer cells. These data demonstrate a novel nonenzymatic function of PLCγ as an important mechanism of plexin-mediated signaling which links tyrosine phosphorylation of plexin-B1 to the regulation of a RhoGEF protein and downstream cellular processes.Mammalian semaphorins were originally identified as axon guidance factors but are now recognized also as important regulators of morphogenesis and homeostasis in various organ systems, including the immune, cardiovascular, and renal systems (3-5, 7, 19, 23, 30, 35, 40, 56, 64, 76). Most effects of semaphorins are mediated by a group of large transmembrane proteins called plexins, of which four families exist in the mammalian system: plexin-A1 to -4, plexin-B1 to -3, plexin-C1, and plexin-D1 (60, 61). The four members of the plexin-A family in most cases require neuropilins as ligand binding partners to respond to semaphorins, whereas the three members of the plexin-B family are directly activated by semaphorins. While plexin-B1 binds Sema4D, plexin-B2 can be activated by Sema4C and Sema4D, and plexin-B3 has been shown to respond to Sema5A (31, 35).The activation of plexins by semaphorins initiates a variety of signaling processes, which involve several small GTPases of the Ras and Rho families (31, 34, 43). All plexin family members possess an R-Ras GTPase-activating protein (GAP) domain (36). Activated plexin-B1 and -A1 have been shown to also interact with other small GTPases, including GTP-bound Rac1 and RhoD as well as Rnd1, Rnd2, and Rnd3 (14, 37, 48, 63, 67, 68, 74). Different from other plexin families, the C terminus of B-family plexins contains a PDZ domain-binding motif which mediates a stable interaction with the guanine nucleotide exchange factors PDZ-RhoGEF and LARG (1, 15, 26, 39, 57). Activation of the plexin-B1/PDZ-RhoGEF complex by semaphorin 4D (Sema4D) results in RhoA activation downstream of plexin-B1 (15, 39, 57). Members of the plexin-B family also interact with and are phosphorylated by the receptor tyrosine kinases ErbB-2 and c-Met (12, 22, 58). ErbB-2-mediated phosphorylation of plexin-B1 is required for plexin-mediated RhoA activation and downstream cellular effects, including the promigratory effects of Sema4D on cancer cells and the induction of axonal growth cone collapse by Sema4D (58, 59). However, the molecular mechanisms linking ErbB-2-mediated phosphorylation of plexin-B1 to the regulation of RhoA activity and subsequent cellular effects are unknown.Here we report that upon activation by Sema4D, plexin-B1 becomes phosphorylated by ErbB-2 at particular tyrosine residues on its intracellular portion. These phosphorylated tyrosine residues serve as docking sites for the SH2 domains of PLCγ. PLCγ is thereby recruited into the plexin-B1 receptor complex and through its SH3 domain mediates RhoA activation and downstream cellular effects.     

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.     

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.