Effects of Phosphorylation of Herpes Simplex Virus 1 Envelope Glycoprotein B by Us3 Kinase In Vivo and In Vitro

We recently reported that the herpes simplex virus 1 (HSV-1) Us3 protein kinase phosphorylates threonine at position 887 (Thr-887) in the cytoplasmic tail of envelope glycoprotein B (gB) (A. Kato, J. Arii, I. Shiratori, H. Akashi, H. Arase, and Y. Kawaguchi, J. Virol. 83:250-261, 2009; T. Wisner, C. C. Wright, A. Kato, Y. Kawaguchi, F. Mou, J. D. Baines, R. J. Roller and D. C. Johnson, J. Virol. 83:3115-3126, 2009). In the studies reported here, we examined the effect(s) of this phosphorylation on viral replication and pathogenesis in vivo and present data showing that replacement of gB Thr-887 by alanine significantly reduced viral replication in the mouse cornea and development of herpes stroma keratitis and periocular skin disease in mice. The same effects have been reported for mice infected with a recombinant HSV-1 carrying a kinase-inactive mutant of Us3. These observations suggested that Us3 phosphorylation of gB Thr-887 played a critical role in viral replication in vivo and in HSV-1 pathogenesis. In addition, we generated a monoclonal antibody that specifically reacted with phosphorylated gB Thr-887 and used this antibody to show that Us3 phosphorylation of gB Thr-887 regulated subcellular localization of gB, particularly on the cell surface of infected cells.The herpes simplex virus 1 (HSV-1) Us3 gene encodes a serine/threonine protein kinase with an amino acid sequence that is conserved in the subfamily Alphaherpesvirinae (9, 20, 29). The Us3 kinase phosphorylation target site has been reported to be similar to that of protein kinase A (PKA), a cellular cyclic AMP-dependent protein kinase (3, 12). Us3 catalytic activity plays important roles in viral replication and pathogenesis in vivo, based on studies showing that recombinant Us3 null mutant viruses and recombinant viruses encoding catalytically inactive Us3 have significantly reduced virulence, pathogenicity, and replication in mouse models (21, 34). In contrast, Us3 is not essential for growth in tissue culture cells (29). Thus, recombinant Us3 mutants grow as well as wild-type virus in Vero cells and have modestly impaired growth in a specific cell line such as HEp-2 cells (32, 33). The catalytic activity of Us3 is, in part, regulated by autophosphorylation of its serine at position 147 (Ser-147), and regulation of Us3 activity by autophosphorylation of Ser-147 appears to play a critical role in HSV-1 replication in vivo and in HSV-1 pathogenesis (34). Numerous studies have elucidated the potential downstream effects of Us3, including blocking apoptosis (18, 26-28), promoting nuclear egress of progeny nucleocapsids through the nuclear membrane (24, 32, 33), redistributing and phosphorylating nuclear membrane-associated viral nuclear egress factors UL31 and UL34 (13, 24, 30, 31) and cellular proteins including lamin A/C and emerin (16, 22, 23), controlling infected cell morphology (12, 27), and downregulating cell surface expression of viral envelope glycoprotein B (gB) (11).Two substrates that mediate some of the Us3 functions described above have been identified. First, it has been shown that Us3 phosphorylates Thr-887 in the cytoplasmic tail of gB, which appears to downregulate cell surface expression of gB (11). This conclusion is based on the observation that a T887A mutation in gB (gB-T887A) markedly upregulated cell surface expression of gB in infected cells: this upregulation was also observed with a recombinant virus encoding a Us3 kinase-inactive mutant, whereas a phosphomimetic substitution for gB Thr-887 restored wild-type cell surface expression of gB (11). Us3 phosphorylation of gB Thr-887 has also been proposed to be involved in regulation of fusion of the nascent progeny virion envelope with the cell''s outer nuclear membrane, based on the observation that virions accumulated aberrantly in the perinuclear space in cells infected with a mutant virus carrying the gB-T887A substitution mutation and lacking the capacity to produce gH (42). Second, it has been shown that Us3 may phosphorylate some or all of the six serines in the UL31 N-terminal region (24). Such phosphorylation might regulate proper localization of UL31 and UL34 at the nuclear membrane, nuclear egress of nucleocapsids, and viral growth in cell cultures since the Us3 kinase-inactive mutant phenotype for nuclear egress (i.e., mislocalization of UL31 and UL34 at the nuclear membrane, aberrant accumulation of virions within herniations of the nuclear membrane, and decreased viral growth in cell cultures) is also produced by replacement of the six serines in the UL31 N-terminal region with alanines while phosphomimetic substitutions of the six serines restored the wild-type phenotype (24).Thus, the molecular mechanisms for some of the downstream effects of Us3 phosphorylation have been gradually elucidated. However, it remains to be shown whether the Us3 functions reported to date are in fact involved in viral replication and pathogenicity in vivo. In the present study, we focused on Us3 phosphorylation of gB Thr-887 and examined the effect(s) of this phosphorylation on viral replication and pathogenesis in vivo. These studies have shown that replacement of gB Thr-887 by alanine significantly reduced viral replication in the mouse cornea and development of herpes stroma keratitis (HSK) and periocular skin disease in mice, as reported for infection of mice with a recombinant virus carrying a Us3 kinase-inactive mutant (34). These observations suggested that Us3 phosphorylation of gB Thr-887 played a critical role in viral replication in vivo and in HSV-1 pathogenesis. In addition, we generated a monoclonal antibody that specifically recognized phosphorylated gB Thr-887 and used this antibody to directly study the functional consequences of Us3 phosphorylation of gB Thr-887 in infected cells. We also present data showing that Us3 phosphorylation of gB Thr-887 regulated subcellular localization of gB, particularly gB localization on the cell surface of infected cells.     

Induction and Inhibition of Type I Interferon Responses by Distinct Components of Lymphocytic Choriomeningitis Virus

Type I interferons (IFNs) play a critical role in the host defense against viruses. Lymphocytic choriomeningitis virus (LCMV) infection induces robust type I IFN production in its natural host, the mouse. However, the mechanisms underlying the induction of type I IFNs in response to LCMV infection have not yet been clearly defined. In the present study, we demonstrate that IRF7 is required for both the early phase (day 1 postinfection) and the late phase (day 2 postinfection) of the type I IFN response to LCMV, and melanoma differentiation-associated gene 5 (MDA5)/mitochondrial antiviral signaling protein (MAVS) signaling is crucial for the late phase of the type I IFN response to LCMV. We further demonstrate that LCMV genomic RNA itself (without other LCMV components) is able to induce type I IFN responses in various cell types by activation of the RNA helicases retinoic acid-inducible gene I (RIG-I) and MDA5. We also show that expression of the LCMV nucleoprotein (NP) inhibits the type I IFN response induced by LCMV RNA and other RIG-I/MDA5 ligands. These virus-host interactions may play important roles in the pathogeneses of LCMV and other human arenavirus diseases.Type I interferons (IFNs), namely, alpha interferon (IFN-α) and IFN-β, are not only essential for host innate defense against viral pathogens but also critically modulate the development of virus-specific adaptive immune responses (6, 8, 28, 30, 36, 50, 61). The importance of type I IFNs in host defense has been demonstrated by studying mice deficient in the type I IFN receptor, which are highly susceptible to most viral pathogens (2, 47, 62).Recent studies have suggested that the production of type I IFNs is controlled by different innate pattern recognition receptors (PRRs) (19, 32, 55, 60). There are three major classes of PRRs, including Toll-like receptors (TLRs) (3, 40), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) (25, 48, 51), and nucleotide oligomerization domain (NOD)-like receptors (9, 22). TLRs are a group of transmembrane proteins expressed on either cell surfaces or endosomal compartments. RLRs localize in the cytosol. Both TLRs and RLRs are involved in detecting viral pathogens and controlling the production of type I IFNs (52, 60). In particular, the endosome-localized TLRs (TLR3, TLR7/8, and TLR9) play important roles in detecting virus-derived double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), and DNA-containing unmethylated CpG motifs, respectively. In contrast, RIG-I detects virus-derived ssRNA with 5′-triphosphates (5′-PPPs) or short dsRNA (<1 kb), whereas melanoma differentiation-associated gene 5 (MDA5) is responsible for recognizing virus-derived long dsRNA as well as a synthetic mimic of viral dsRNA poly(I):poly(C) [poly(I·C)] (24, 60). Recognition of viral pathogen-associated molecular patterns (PAMPs) ultimately leads to the activation and nuclear translocation of interferon regulatory factors (IRFs) and nuclear factor κB (NF-κB), which, in turn, switches on a cascade of genes controlling the production of both type I IFNs and other proinflammatory cytokines (10, 11, 60).Lymphocytic choriomeningitis virus (LCMV) infection in its natural host, the mouse, is an excellent system to study the impact of virus-host interactions on viral pathogenesis and to address important issues related to human viral diseases (1, 45, 49, 67). LCMV infection induces type I IFNs as well as other proinflammatory chemokines and cytokines (6, 41). Our previous studies have demonstrated that TLR2, TLR6, and CD14 are involved in LCMV-induced proinflammatory chemokines and cytokines (66). The mechanism by which LCMV induces type I IFN responses, however, has not been clearly defined (7, 8, 31, 44). The role of the helicase family members RIG-I and MDA5 in virus-induced type I IFN responses has been recently established. RIG-I has been found to be critical in controlling the production of type I IFN in response to a number of RNA viruses, including influenza virus, rabies virus, Hantaan virus, vesicular stomatitis virus (VSV), Sendai virus (SeV), etc. In contrast, MDA5 is required for responses to picornaviruses (15, 25, 63).In the present study, we demonstrated that LCMV genomic RNA strongly activates type I IFNs through a RIG-I/MDA5-dependent signaling pathway. Our present study further demonstrated that the LCMV nucleoprotein (NP) blocks LCMV RNA- and other viral ligand-induced type I IFN responses.     

Mcl-1 Integrates the Opposing Actions of Signaling Pathways That Mediate Survival and Apoptosis

Mcl-1 is a member of the Bcl2-related protein family that is a critical mediator of cell survival. Exposure of cells to stress causes inhibition of Mcl-1 mRNA translation and rapid destruction of Mcl-1 protein by proteasomal degradation mediated by a phosphodegron created by glycogen synthase kinase 3 (GSK3) phosphorylation of Mcl-1. Here we demonstrate that prior phosphorylation of Mcl-1 by the c-Jun N-terminal protein kinase (JNK) is essential for Mcl-1 phosphorylation by GSK3. Stress-induced Mcl-1 degradation therefore requires the coordinated activity of JNK and GSK3. Together, these data establish that Mcl-1 functions as a site of signal integration between the proapoptotic activity of JNK and the prosurvival activity of the AKT pathway that inhibits GSK3.Mcl-1 is an antiapoptotic member of the Bcl2 family. Gene knockout studies of mice demonstrate that Mcl-1 is essential for embryonic development and for the survival of hematopoietic cells (28-30). Studies of the stress response have demonstrated that Mcl-1 plays an important role in the sensitization of cells to apoptotic signals (1, 11, 25). Thus, exposure to UV radiation causes the rapid degradation of Mcl-1 and the release of proapoptotic partner proteins from Mcl-1 complexes (e.g., Bim). The mechanism of rapid Mcl-1 destruction is mediated by the combined actions of two different pathways. First, the exposure to stress causes phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2α) on the inhibitory site Ser-51 that prevents translation of Mcl-1 mRNA (1, 11, 25). Second, Mcl-1 is rapidly degraded by the ubiquitin-dependent proteasome pathway (27). Together, these pathways cause a rapid reduction in Mcl-1 expression. This loss of Mcl-1 may be a required initial response for the apoptosis of cells exposed to stress (25).The E3 ubiquitin protein ligase Mule/ARF-BP1 contains a BH3 domain that interacts with Mcl-1 and can initiate ubiquitin-dependent degradation of Mcl-1 (39). Recent studies have demonstrated that rapid stress-induced degradation of Mcl-1 is mediated by an alternative pathway involving the E3 ubiquitin protein ligase β-TrCP, which binds a stress-induced phosphodegron created by the phosphorylation of Mcl-1 by glycogen synthase kinase 3 (GSK3) (7, 21). How the exposure to stress causes GSK3-mediated phosphorylation of Mcl-1 is unclear, but GSK3 has been shown to directly phosphorylate Mcl-1 (7, 21). Mcl-1 phosphorylation and degradation may therefore be controlled by the prosurvival AKT pathway, which can negatively regulate GSK3 (7, 21).Mcl-1 is critically involved in the regulation of cell survival and is therefore subject to regulation by multiple mechanisms (26). Thus, Mcl-1 gene expression is regulated by many growth factors and cytokines (26), and Mcl-1 mRNA is regulated by microRNA pathways (24). The Mcl-1 protein is stabilized by binding TCTP (20) and the BH3-only protein Bim (4). In contrast, the BH3-only protein Noxa binds and destabilizes Mcl-1 (4, 36). Moreover, it is established that Mcl-1 is phosphorylated by several protein kinases on sites that may regulate Mcl-1 function. Phosphorylation of human Mcl-1 (hMcl-1) on Ser-64 (a site that is not conserved in other species) may enhance antiapoptotic activity by increasing the interaction of Mcl-1 with Bim, Noxa, and Bak (18). Phosphorylation on Ser-121 and Thr-163 may inhibit the antiapoptotic activity of hMcl-1 (15), and phosphorylation on Thr-163 may increase hMcl-1 protein stability (9). The conserved GSK3 phosphorylation site Ser-159 (and possibly Ser-155) can initiate rapid proteasomal degradation of hMcl-1 (7, 21). Together, these findings suggest that the function of Mcl-1 is very tightly regulated.The results of previous studies have implicated the c-Jun N-terminal protein kinase (JNK) in the regulation of Mcl-1 (15, 18). The purpose of this study was to test whether Mcl-1 is a target of signal transduction by JNK. We demonstrate that a key function of JNK is to prime Mcl-1 for phosphorylation by GSK3. JNK is required for GSK3-mediated degradation of Mcl-1 in response to stress. Coordinated regulation of the stress-activated JNK pathway and the AKT-inhibited GSK3 pathway is therefore required for stress-induced Mcl-1 degradation.     

mTORC1-Activated S6K1 Phosphorylates Rictor on Threonine 1135 and Regulates mTORC2 Signaling

The mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that forms two functionally distinct complexes important for nutrient and growth factor signaling. While mTOR complex 1 (mTORC1) regulates mRNA translation and ribosome biogenesis, mTORC2 plays an important role in the phosphorylation and subsequent activation of Akt. Interestingly, mTORC1 negatively regulates Akt activation, but whether mTORC1 signaling directly targets mTORC2 remains unknown. Here we show that growth factors promote the phosphorylation of Rictor (rapamycin-insensitive companion of mTOR), an essential subunit of mTORC2. We found that Rictor phosphorylation requires mTORC1 activity and, more specifically, the p70 ribosomal S6 kinase 1 (S6K1). We identified several phosphorylation sites in Rictor and found that Thr1135 is directly phosphorylated by S6K1 in vitro and in vivo, in a rapamycin-sensitive manner. Phosphorylation of Rictor on Thr1135 did not affect mTORC2 assembly, kinase activity, or cellular localization. However, cells expressing a Rictor T1135A mutant were found to have increased mTORC2-dependent phosphorylation of Akt. In addition, phosphorylation of the Akt substrates FoxO1/3a and glycogen synthase kinase 3α/β (GSK3α/β) was found to be increased in these cells, indicating that S6K1-mediated phosphorylation of Rictor inhibits mTORC2 and Akt signaling. Together, our results uncover a new regulatory link between the two mTOR complexes, whereby Rictor integrates mTORC1-dependent signaling.The mammalian target of rapamycin (mTOR) is an evolutionarily conserved phosphatidylinositol 3-kinase (PI3K)-related Ser/Thr kinase that integrates signals from nutrients, energy sufficiency, and growth factors to regulate cell growth as well as organ and body size in a variety of organisms (reviewed in references 4, 38, 49, and 77). mTOR was discovered as the molecular target of rapamycin, an antifungal agent used clinically as an immunosuppressant and more recently as an anticancer drug (5, 20). Recent evidence indicates that deregulation of the mTOR pathway occurs in a majority of human cancers (12, 18, 25, 46), suggesting that rapamycin analogs may be potent antineoplastic therapeutic agents.mTOR forms two distinct multiprotein complexes, the rapamycin-sensitive and -insensitive mTOR complexes 1 and 2 (mTORC1 and mTORC2), respectively (6, 47). In cells, rapamycin interacts with FKBP12 and targets the FKBP12-rapamycin binding (FRB) domain of mTORC1, thereby inhibiting some of its function (13, 40, 66). mTORC1 is comprised of the mTOR catalytic subunit and four associated proteins, Raptor (regulatory associated protein of mTOR), mLST8 (mammalian lethal with sec13 protein 8), PRAS40 (proline-rich Akt substrate of 40 kDa), and Deptor (28, 43, 44, 47, 59, 73, 74). The small GTPase Rheb (Ras homolog enriched in brain) is a key upstream activator of mTORC1 that is negatively regulated by the tuberous sclerosis complex 1 (TSC1)/TSC2 GTPase-activating protein complex (reviewed in reference 35). mTORC1 is activated by PI3K and Ras signaling through direct phosphorylation and inactivation of TSC2 by Akt, extracellular signal-regulated kinase (ERK), and p90 ribosomal protein S6 kinase (RSK) (11, 37, 48, 53, 63). mTORC1 activity is also regulated at the level of Raptor. Whereas low cellular energy levels negatively regulate mTORC1 activity through phosphorylation of Raptor by AMP-activated protein kinase (AMPK) (27), growth signaling pathways activating the Ras/ERK pathway positively regulate mTORC1 activity through direct phosphorylation of Raptor by RSK (10). More recent evidence has also shown that mTOR itself positively regulates mTORC1 activity by directly phosphorylating Raptor at proline-directed sites (20a, 75). Countertransport of amino acids (55) and amino acid signaling through the Rag GTPases were also shown to regulate mTORC1 activity (45, 65). When activated, mTORC1 phosphorylates two main regulators of mRNA translation and ribosome biogenesis, the AGC (protein kinase A, G, and C) family kinase p70 ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and thus stimulates protein synthesis and cellular growth (50, 60).The second mTOR complex, mTORC2, is comprised of mTOR, Rictor (rapamycin-insensitive companion of mTOR), mSin1 (mammalian stress-activated mitogen-activated protein kinase-interacting protein 1), mLST8, PRR5 (proline-rich region 5), and Deptor (21, 39, 58, 59, 66, 76, 79). Rapamycin does not directly target and inhibit mTORC2, but long-term treatment with this drug was shown to correlate with mTORC2 disassembly and cytoplasmic accumulation of Rictor (21, 39, 62, 79). Whereas mTORC1 regulates hydrophobic motif phosphorylation of S6K1, mTORC2 has been shown to phosphorylate other members of the AGC family of kinases. Biochemical and genetic evidence has demonstrated that mTORC2 phosphorylates Akt at Ser473 (26, 39, 68, 70), thereby contributing to growth factor-mediated Akt activation (6, 7, 52). Deletion or knockdown of the mTORC2 components mTOR, Rictor, mSin1, and mLST8 has a dramatic effect on mTORC2 assembly and Akt phosphorylation at Ser473 (26, 39, 79). mTORC2 was also shown to regulate protein kinase Cα (PKCα) (26, 66) and, more recently, serum- and glucocorticoid-induced protein kinase 1 (SGK1) (4, 22). Recent evidence implicates mTORC2 in the regulation of Akt and PKCα phosphorylation at their turn motifs (19, 36), but whether mTOR directly phosphorylates these sites remains a subject of debate (4).Activation of mTORC1 has been shown to negatively regulate Akt phosphorylation in response to insulin or insulin-like growth factor 1 (IGF1) (reviewed in references 30 and 51). This negative regulation is particularly evident in cell culture models with defects in the TSC1/TSC2 complex, where mTORC1 and S6K1 are constitutively activated. Phosphorylation of insulin receptor substrate-1 (IRS-1) by mTORC1 (72) and its downstream target S6K1 has been shown to decrease its stability and lead to an inability of insulin or IGF1 to activate PI3K and Akt (29, 69). Although the mechanism is unknown, platelet-derived growth factor receptor β (PDGF-Rβ) has been found to be downregulated in TSC1- and TSC2-deficient murine embryonic fibroblasts (MEFs), contributing to a reduction of PI3K signaling (80). Interestingly, inhibition of Akt phosphorylation by mTORC1 has also been observed in the presence of growth factors other than IGF-1, insulin, or PDGF, suggesting that there are other mechanisms by which mTORC1 activation restricts Akt activity in cells (reviewed in references 6 and 31). Recent evidence demonstrates that rapamycin treatment causes a significant increase in Rictor electrophoretic mobility (2, 62), suggesting that phosphorylation of the mTORC2 subunit Rictor may be regulated by mTORC1 or downstream protein kinases.Herein, we demonstrate that Rictor is phosphorylated by S6K1 in response to mTORC1 activation. We demonstrate that Thr1135 is directly phosphorylated by S6K1 and found that a Rictor mutant lacking this phosphorylation site increases Akt phosphorylation induced by growth factor stimulation. Cells expressing the Rictor T1135A mutant were found to have increased Akt signaling to its substrates compared to Rictor wild-type- and T1135D mutant-expressing cells. Together, our results suggest that Rictor integrates mTORC1 signaling via its phosphorylation by S6K1, resulting in the inhibition of mTORC2 and Akt signaling.     

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.     

Extracellular Signal-Regulated Kinase Promotes Rho-Dependent Focal Adhesion Formation by Suppressing p190A RhoGAP

Cell migration is critical for normal development and for pathological processes including cancer cell metastasis. Dynamic remodeling of focal adhesions and the actin cytoskeleton are crucial determinants of cell motility. The Rho family and the mitogen-activated protein kinase (MAPK) module consisting of MEK-extracellular signal-regulated kinase (ERK) are important regulators of these processes, but mechanisms for the integration of these signals during spreading and motility are incompletely understood. Here we show that ERK activity is required for fibronectin-stimulated Rho-GTP loading, Rho-kinase function, and the maturation of focal adhesions in spreading cells. We identify p190A RhoGAP as a major target for ERK signaling in adhesion assembly and identify roles for ERK phosphorylation of the C terminus in p190A localization and activity. These observations reveal a novel role for ERK signaling in adhesion assembly in addition to its established role in adhesion disassembly.Cell migration is a highly coordinated process essential for physiological and pathological processes (69). Signaling through Rho family GTPases (e.g., Rac, Cdc42, and Rho) is crucial for cell migration. Activated Rac and Cdc42 are involved in the production of a dominant lamellipodium and filopodia, respectively, whereas Rho-stimulated contractile forces are required for tail retraction and to maintain adhesion to the matrix (57, 58, 68). Rac- and Cdc42-dependent membrane protrusions are driven by the actin cytoskeleton and the formation of peripheral focal complexes; Rho activation stabilizes protrusions by stimulating the formation of mature focal adhesions and stress fibers. Active Rho influences cytoskeletal dynamics through effectors including the Rho kinases (ROCKs) (2, 3).Rho activity is stimulated by GEFs that promote GTP binding and attenuated by GTPase-activating proteins (GAPs) that enhance Rho''s intrinsic GTPase activity. However, due to the large number of RhoGEFs and RhoGAPs expressed in mammalian cells, the molecular mechanisms responsible for regulation of Rho activity in time and space are incompletely understood. p190A RhoGAP (hereafter p190A) is implicated in adhesion and migration signaling. p190A contains an N-terminal GTPase domain, a large middle domain juxtaposed to the C-terminal GAP domain, and a short C-terminal tail (74). The C-terminal tail of ∼50 amino acids is divergent between p190A and the closely related family member p190B (14) and thus may specify the unique functional roles for p190A and p190B revealed in gene knockout studies (10, 11, 41, 77, 78). p190A activity is dynamically regulated in response to external cues during cell adhesion and migration (5, 6, 59). Arthur et al. (5) reported that p190A activity is required for the transient decrease in RhoGTP levels seen in fibroblasts adhering to fibronectin. p190A activity is positively regulated by tyrosine phosphorylation (4, 5, 8, 17, 31, 39, 40, 42): phosphorylation at Y1105 promotes its association with p120RasGAP and subsequent recruitment to membranes or cytoskeleton (8, 17, 27, 31, 71, 75, 84). However, Y1105 phosphorylation is alone insufficient to activate p190A GAP activity (39). While the functions of p190A can be irreversibly terminated by ubiquitinylation in a cell-cycle-dependent manner (80), less is known about reversible mechanisms that negatively regulate p190A GAP activity during adhesion and motility.The integration of Rho family GTPase and extracellular signal-regulated kinase (ERK) signaling is important for cell motility (48, 50, 63, 76, 79). Several studies have demonstrated a requirement for ERK signaling in the disassembly of focal adhesions in migrating cells, in part through the activation of calpain proteases (36, 37) that can downregulate focal adhesion kinase (FAK) signaling (15), locally suppress Rho activity (52), and sever cytoskeletal linkers to focal adhesions (7, 33). Inhibition of ERK signaling increases focal adhesion size and retards disassembly of focal adhesions in adherent cells (57, 64, 85, 86). It is also recognized that ERK modulates Rho-dependent cellular processes, including membrane protrusion and migration (18, 25, 64, 86). Interestingly, ERK activated in response to acute fibronectin stimulation localizes not only to mature focal adhesions, but also to peripheral focal complexes (32, 76). Since these complexes can either mature or be turned over (12), ERK may play a distinct role in focal adhesion assembly. ERK is proposed to promote focal adhesion formation by activating myosin light chain kinase (MLCK) (21, 32, 50).Here we find that ERK activity is required for Rho activation and focal adhesion formation during adhesion to fibronectin and that p190A is an essential target of ERK signaling in this context. Inspection of the p190A C terminus reveals a number of consensus ERK sites and indeed p190A is phosphorylated by recombinant ERK only on its C terminus in vitro, and on the same C-terminal peptide in vivo. Mutation of the C-terminal ERK phosphorylation sites to alanine increases the biochemical and biological activity of p190A. Finally, inhibition of MEK or mutation of the C-terminal phosphorylation sites enhances retention of p190A in peripheral membranes during spreading on fibronectin. Our data support the conclusion that ERK phosphorylation inhibits p190A allowing increases in RhoGTP and cytoskeletal changes necessary for focal adhesion formation.