Src phosphorylates ezrin at tyrosine 477 and induces a phosphospecific association between ezrin and a kelch-repeat protein family member

Ezrin, a linker between plasma membrane and actin cytoskeleton possesses morphogenic properties and can promote dissemination of tumor cells. Ezrin is phosphorylated on tyrosine, but a detailed picture of the signaling pathways involved in this modification is lacking. The transforming tyrosine kinase Src has various cytoskeletal substrates and is involved in regulation of cellular adhesion. We studied the role of Src in tyrosine phosphorylation of ezrin in adherent cells. We show that ezrin is phosphorylated in human embryonic kidney 293 cells in a Src family-dependent way. In SYF cells lacking Src, Yes, and Fyn, ezrin was not tyrosine-phosphorylated but reintroduction of wild-type Src followed by Src activation or introduction of active Src restored phosphorylation. Mapping of the Src-catalyzed tyrosine in vitro and in vivo by site-directed mutagenesis demonstrated Tyr(477) as the primary target residue. We generated a pTyr(477)-phosphospecific antibody, which confirmed that Tyr(477) becomes phosphorylated in cells in a Src-dependent manner. Tyr(477) phosphorylation did not affect ezrin head-to-tail association or phosphorylation of ezrin on threonine 566, indicating that the function of Tyr(477) phosphorylation is not related to the intramolecular regulation of ezrin. A modified yeast two-hybrid screen in which ezrin bait was phosphorylated by Src identified a novel interaction with a kelch-repeat protein family member, KBTBD2 (Kelch-repeat and BTB/POZ domain containing 2). The Src dependence of the interaction was further verified by affinity precipitation assays. Identification of a functional interplay with Src opens novel avenues for further characterization of the biological activities of ezrin.     

Tyrosine phosphorylation of the alpha subunit of transducin and its association with Src in photoreceptor rod outer segments

Recent evidence indicates that tyrosine phosphorylation may play important roles in retinal photoreceptor rod outer segments (ROS). We investigated the tyrosine phosphorylation of endogenous proteins in isolated bovine ROS. Several proteins with apparent molecular masses of 31, 39, 60, 83, 90, 97, 120, 140, and 180 kDa were tyrosine-phosphorylated in ROS incubated with Mg(2+), ATP, and orthovanadate. Several tyrosine kinase inhibitors significantly inhibited tyrosine phosphorylation of these proteins in ROS. The 39- and 60-kDa tyrosine-phosphorylated proteins were identified as the alpha subunit of the G protein transducin (Talpha) and the tyrosine kinase Src, respectively. The presence of Src and tyrosine kinase activity in bovine ROS was confirmed by their cofractionation with rhodopsin and Talpha on continuous sucrose gradients. Several tyrosine-phosphorylated proteins, including Src, coimmunoprecipitated with Talpha. The association of Src with Talpha was detected in the absence of tyrosine phosphorylation, but was enhanced with increased tyrosine phosphorylation of ROS. Moreover, tyrosine kinase activity also associated with Talpha was sevenfold higher under tyrosine-phosphorylating conditions. The recovery of transducin by hypotonic GTP extraction from tyrosine-phosphorylated ROS was significantly less than that from nonphosphorylated ROS. We localized the site on Talpha phosphorylated by Src to the amino-terminal half by limited tryptic digests, and further mapped it by ion trap mass spectrometry to Tyr(142) in the helical domain of Talpha. Talpha was also tyrosine-phosphorylated in vivo in rat retina, but this phosphorylation was not affected by light.     

Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK.

Changes in cellular growth and dramatic alterations in cell morphology and adhesion are common features of cells transformed by oncogenic protein tyrosine kinases, such as pp60src and other members of the Src family. In this report, we present evidence for the stable association of two Src family kinases (pp60src and pp59fyn) with tyrosine-phosphorylated forms of a focal adhesion-associated protein tyrosine kinase, pp125FAK. In Src-transformed chicken embryo cells, most of the pp125FAK was stably complexed with activated pp60src (e.g., pp60(527F). The stable association of pp125FAK with pp60(527F) in vivo required the structural integrity of the Src SH2 domain. The association of pp60(527F) and pp125FAK could be reconstituted in vitro by incubation of normal cell extracts with glutathione S-transferase fusion proteins containing SH2 or SH3/SH2 domains of pp60src. Furthermore, the association of isolated SH2 or SH3/SH2 domains with in vitro 32P-labeled pp125FAK protected the major site of pp125FAK autophosphorylation from digestion with a tyrosine phosphatase, indicating that the autophosphorylation site of pp125FAK participates in binding with Src. Immunoprecipitation of Src family kinases from extracts of normal chicken embryo cells revealed stable complexes of pp59fyn and tyrosine-phosphorylated pp125FAK. These data provide evidence for a direct interaction between two cytoplasmic nonreceptor protein tyrosine kinases and suggest that Src may contribute to changes in pp125FAK regulation in transformed cells. Furthermore, pp125FAK may directly participate in the targeting of pp59fyn or possibly other Src family kinases to focal adhesions in normal cells.     

Localization of PTP-1B, SHP-2, and Src exclusively in rat brain mitochondria and functional consequences

An immunodetection study of protein tyrosine phosphatase 1B (PTP-1B), SHP-2, and Src in isolated mitochondria from different rat tissues (brain, muscle, heart, liver, and kidney) revealed their exclusive localization in the brain. Given this result, we sought whether mitochondria respond to ATP and to the general tyrosine phosphatase inhibitor orthovanadate and found little or no change in the tyrosine phosphorylation profile of mitochondria from muscle, heart, liver, and kidney. In contrast, ATP induced an enhancement in the tyrosine-phosphorylated protein profile of brain mitochondria, which was further greatly enhanced with orthovanadate and which disappeared when Src was inhibited with two inhibitors: PP2 and PP1. Importantly, we found that in brain mitochondria, ATP addition induced Src autophosphorylation at Tyr-416 in its catalytic site, leading to its activation, whereas the regulatory Tyr-527 site remained unphosphorylated. Functional implications were addressed by measurements of the enzymatic activity of each of the oxidative phosphorylation complexes in brain mitochondria in the presence of ATP. We found an increase in complex I, III, and IV activity and a decrease in complex V activity, partially reversed by Src inhibition, demonstrating that the complexes are Src substrates. These results complemented and reinforced our initial study showing that respiration of brain mitochondria was partially dependent on tyrosine phosphorylation. Therefore, the present data suggest a possible control point in the regulation of respiration by tyrosine phosphorylation of the complexes mediated by Src auto-activation.     

Gi-mediated activation of the Ras/MAP kinase pathway involves a 100 kDa tyrosine-phosphorylated Grb2 SH3 binding protein, but not Src nor Shc.

Mitogenic G protein-coupled receptors, such as those for lysophosphatidic acid (LPA) and thrombin, activate the Ras/MAP kinase pathway via pertussis toxin (PTX)-sensitive Gi, tyrosine kinase activity and recruitment of Grb2, which targets guanine nucleotide exchange activity to Ras. Little is known about the tyrosine phosphorylations involved, although Src activation and Shc phosphorylation are thought to be critical. We find that agonist-induced Src activation in Rat-1 cells is not mediated by Gi and shows no correlation with Ras/MAP kinase activation. Furthermore, LPA-induced tyrosine phosphorylation of Shc is PTX-insensitive and Ca2+-dependent in COS cells, but undetectable in Rat-1 cells. Expression of dominant-negative Src or Shc does not affect MAP kinase activation by LPA. Thus, Gi-mediated Ras/MAP kinase activation in fibroblasts and COS cells involves neither Src nor Shc. Instead, we detect a 100 kDa tyrosine-phosphorylated protein (p100) that binds to the C-terminal SH3 domain of Grb2 in a strictly Gi- and agonist-dependent manner. Tyrosine kinase inhibitors and wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, prevent p100-Grb2 complex formation and MAP kinase activation by LPA. Our results suggest that the p100-Grb2 complex, together with an upstream non-Src tyrosine kinase and PI 3-kinase, couples Gi to Ras/MAP kinase activation, while Src and Shc act in a different pathway.     

Interaction of the SH2 domain of Fyn with a cytoskeletal protein, beta-adducin

Fyn is a Src family tyrosine kinase expressed abundantly in neurons and believed to have specific functions in the brain. To understand the function of Fyn tyrosine kinase, we attempted to identify Fyn Src homology 2 (SH2) domain-binding proteins from a Nonidet P-40-insoluble fraction of the mouse brain. beta-Adducin, an actin filament-associated cytoskeletal protein, was isolated by two-dimensional gel electrophoresis and identified by tandem mass spectrometry. beta-Adducin was tyrosine phosphorylated by coexpression with wild type but not with a kinase-negative form of Fyn in COS-7 cells. Cell staining analysis showed that coexpression of beta-adducin with Fyn induced translocation of beta-adducin from the cytoplasm to the periphery of the cells where it was colocalized with actin filaments and Fyn. These findings suggest that tyrosine-phosphorylated beta-adducin associates with the SH2 domain of Fyn and colocalizes under plasma membranes.     

CD44-initiated cell spreading induces Pyk2 phosphorylation, is mediated by Src family kinases, and is negatively regulated by CD45

CD44 is a cell adhesion molecule implicated in leukocyte adhesion and migration, co-stimulation of T cells, and tumor metastasis. CD45 is a leukocyte-specific protein tyrosine phosphatase that dephosphorylates the Src family kinases, Lck and Fyn, in T cells. Positive regulation of Lck by CD45 is required for its effective participation in T cell receptor signaling events. Here, immobilized CD44 antibody induced a distinctive cell spreading in CD45(-), but not CD45(+), T cells, and this correlated with the induction of tyrosine-phosphorylated proteins. Two focal adhesion family kinases, Pyk2 and, to a lesser extent, FAK were inducibly phosphorylated, as was a potential substrate, Cas. CD44-mediated cell spreading and induced tyrosine phosphorylation were prevented by the Src family kinase inhibitor, PP2. Furthermore, 2-fold more Lck associated with CD44 in the low density sucrose fraction from CD45(-) T cells compared with CD45(+) T cells, suggesting that CD45 may regulate the association of Lck with CD44 in this fraction. Therefore, in CD45(-) T cells, CD44 signaling is mediated by Src family kinases, and this leads to Pyk2 phosphorylation, cytoskeletal changes, and cell spreading. This implicates CD45 in the negative regulation of Src family kinase-mediated CD44 signaling leading to T cell spreading.     

Evidence of a role for SHP-1 in platelet activation by the collagen receptor glycoprotein VI

The Src homology (SH)2 domain-containing protein-tyrosine phosphatase SHP-1 is tyrosine phosphorylated in platelets in response to the glycoprotein VI (GPVI)-selective agonist collagen-related peptide (CRP), collagen, and thrombin. Two major unidentified tyrosine-phosphorylated bands of 28 and 32 kDa and a minor band of 130 kDa coprecipitate with SHP-1 in response to all three agonists. Additionally, tyrosine-phosphorylated proteins of 50-55 and 70 kDa specifically associate with SHP-1 following stimulation by CRP and collagen. The tyrosine kinases Lyn, which exists as a 53 and 56-kDa doublet, and Syk were identified as major components of these bands, respectively. Kinase assays on SHP-1 immunoprecipitates performed in the presence of the Src family kinase inhibitor PP1 confirmed the presence of a Src kinase in CRP- but not thrombin-stimulated cells. Lyn, Syk, and SLP-76, along with tyrosine-phosphorylated 28-, 32-, and 130-kDa proteins, bound selectively to a glutathione S-transferase protein encoding the SH2 domains of SHP-1, suggesting that this is the major site of interaction. Platelets isolated from motheaten viable mice (mev/mev) revealed the presence of a heavily tyrosine-phosphorylated 26-kDa protein that was not found in wild-type platelets. CRP-stimulated mev/mev platelets manifested hypophosphorylation of Syk and Lyn and reduced P-selectin expression relative to controls. These observations provide evidence of a functional role for SHP-1 in platelet activation by GPVI.     

Activation of Src kinase in skeletal muscle cells by 1, 1,25-(OH(2))-vitamin D(3) correlates with tyrosine phosphorylation of the vitamin D receptor (VDR) and VDR-Src interaction

The rapid effect of 1 alpha,25(OH(2))-vitamin D(3) [1 alpha, 25(OH(2))D(3)] on tyrosine kinase Src and its relationship to the vitamin D receptor (VDR) was investigated to further characterize the hormone signaling mechanism in chick muscle cells. Exposure of cultured myotubes to 1 alpha,25(OH(2))D(3) caused a time-dependent increase in Src activity, which was evident at 1 min (one-fold) and reached a maximum at 5 min (15-fold). Immunoblotting with anti-phosphotyrosine antibody of immunoprecipitated Src showed that the hormone decreased Src tyrosine phosphorylation state with maximal effects at 5 min. Using a database for protein consensus motifs we found a putative tyrosine phosphorylation site (amino acids 164-170: KTFDTTY) within the primary sequence of the chick VDR. When the myotube VDR was immunoprecipitated it appeared onto SDS-PAGE gels as a single band of 58 kDa recognized by an anti-phosphotyrosine antibody. Prior treatment of cells with (1)alpha,25(OH(2))D(3) significantly increased tyrosine phosphorylation of the VDR (two- to three-fold above basal levels). In agreement with Src being a SH2-domain containing protein involved in recognition of tyrosine-phosphorylated targets, immunoprecipitation with anti-Src antibody under native conditions followed by blotting with anti-VDR antibody, or using the antibodies in inverse order, showed that the VDR co-precipitates with Src, thus indicating the existence of a VDR/Src complex. Stimulation with the cognate VDR ligand significantly increased formation of the complex with respect to basal conditions. These results altogether provide the first evidence to date for 1 alpha,25(OH(2))D(3) activation involving Src association to tyrosine phosphorylated VDR.     

Cortactin and Crk cooperate to trigger actin polymerization during Shigella invasion of epithelial cells

Shigella, the causative agent of bacillary dysentery, invades epithelial cells in a process involving Src tyrosine kinase signaling. Cortactin, a ubiquitous actin-binding protein present in structures of dynamic actin assembly, is the major protein tyrosine phosphorylated during Shigella invasion. Here, we report that RNA interference silencing of cortactin expression, as does Src inhibition in cells expressing kinase-inactive Src, interferes with actin polymerization required for the formation of cellular extensions engulfing the bacteria. Shigella invasion induced the recruitment of cortactin at plasma membranes in a tyrosine phosphorylation-dependent manner. Overexpression of wild-type forms of cortactin or the adaptor protein Crk favored Shigella uptake, and Arp2/3 binding-deficient cortactin derivatives or an Src homology 2 domain Crk mutant interfered with bacterial-induced actin foci formation. Crk was shown to directly interact with tyrosine-phosphorylated cortactin and to condition cortactin-dependent actin polymerization required for Shigella uptake. These results point at a major role for a Crk-cortactin complex in actin polymerization downstream of tyrosine kinase signaling.     

Tyrosine-phosphorylated low density lipoprotein receptor-related protein 1 (Lrp1) associates with the adaptor protein SHC in SRC-transformed cells

v-Src transforms fibroblasts in vitro and causes tumor formation in the animal by tyrosine phosphorylation of critical cellular substrates. Exactly how v-Src interacts with these substrates remains unknown. One of its substrates, the adaptor protein Shc, is thought to play a crucial role during cellular transformation by v-Src by linking v-Src to Ras. We used Shc proteins with mutations in either the phosphotyrosine binding (PTB) or Src homology 2 domain to determine that phosphorylation of Shc in v-Src-expressing cells depends on the presence of a functional PTB domain. We purified a 100-kDa Shc PTB-binding protein from Src-transformed cells that was identified as the beta chain of the low density lipoprotein receptor-related protein LRP1. LRP1 acts as an import receptor for a variety of proteins and is involved in clearance of the beta-amyloid precursor protein. This study shows that LRP1 is tyrosine-phosphorylated in v-Src-transformed cells and that tyrosine-phosphorylated LRP1 binds in vivo and in vitro to Shc. The association between Shc and LRP1 may provide a mechanism for recruitment of Shc to the plasma membrane where it is phosphorylated by v-Src. It is at the membrane that Shc is thought to be involved in Ras activation. These observations further suggest that LRP1 could function as a signaling receptor and may provide new avenues to investigate its possible role during embryonal development and the onset of Alzheimer's disease.     

The mixed-lineage kinase DLK undergoes Src-dependent tyrosine phosphorylation and activation in cells exposed to vanadate or platelet-derived growth factor (PDGF)

Some data in the literature suggest that serine/threonine phosphorylation is required for activation of the mixed-lineage kinases (MLKs), a subgroup of mitogen-activated protein kinase kinase kinases (MAPKKKs). In this report, we demonstrate that the MLK family member DLK is activated and concurrently tyrosine-phosphorylated in cells exposed to the protein tyrosine phosphatase inhibitor vanadate. Tyrosine phosphorylation appears crucial for activation as incubation of vanadate-activated DLK molecules with a tyrosine phosphatase substantially reduced DLK enzymatic activity. Interestingly, the effects of vanadate on DLK are completely blocked by treatment with a Src family kinase inhibitor, PP2, or the expression of short hairpin RNA (shRNA) directed against Src. DLK also fails to undergo vanadate-stimulated tyrosine phosphorylation and activation in fibroblasts which lack expression of Src, Yes and Fyn, but reintroduction of wild-type Src or Fyn followed by vanadate treatment restores this response. In addition to vanadate, stimulation of cells with platelet-derived growth factor (PDGF) also induces tyrosine phosphorylation and activation of DLK by a Src-dependent mechanism. DLK seems important for PDGF signaling because its depletion by RNA interference substantially reduces PDGF-stimulated ERK and Akt kinase activation. Thus, our findings suggest that Src-dependent tyrosine phosphorylation of DLK may be important for regulation of its activity, and they support a role for DLK in PDGF signaling.     

Tyrosine-phosphorylated caveolin is a physiological substrate of the low M(r) protein-tyrosine phosphatase

Low M(r) phosphotyrosine-protein phosphatase is involved in the regulation of several tyrosine kinase growth factor receptors. The best characterized action of this enzyme is on the signaling pathways activated by platelet-derived growth factor, where it plays multiple roles. In this study we identify tyrosine-phosphorylated caveolin as a new potential substrate for low M(r) phosphotyrosine-protein phosphatase. Caveolin is tyrosine-phosphorylated in vivo by Src kinases, recruits into caveolae, and hence regulates the activities of several proteins involved in cellular signaling cascades. Our results demonstrate that caveolin and low M(r) phosphotyrosine-protein phosphatase coimmunoprecipitate from cell lysates, and that a fraction of the enzyme localizes in caveolae. Furthermore, in a cell line sensitive to insulin, the overexpression of the C12S dominant negative mutant of low M(r) phosphotyrosine-protein phosphatase (a form lacking activity but able to bind substrates) causes the enhancement of tyrosine-phosphorylated caveolin. Insulin stimulation of these cells induces a strong increase of caveolin phosphorylation. The localization of low M(r) phosphotyrosine-protein phosphatase in caveolae, the in vivo interaction between this enzyme and caveolin, and the capacity of this enzyme to rapidly dephosphorylate phosphocaveolin, all indicate that tyrosine-phosphorylated caveolin is a relevant substrate for this phosphatase.     

Identification and cloning of xp95, a putative signal transduction protein in Xenopus oocytes

A 95-kDa protein in Xenopus oocytes, Xp95, was shown to be phosphorylated from the first through the second meiotic divisions during progesterone-induced oocyte maturation. Xp95 was purified and cloned. The Xp95 protein sequence exhibited homology to mouse Rhophilin, budding yeast Bro1, and Aspergillus PalA, all of which are implicated in signal transduction. It also contained three conserved features including seven conserved tyrosines, a phosphorylation consensus sequence for the Src family of tyrosine kinases, and a proline-rich domain near the C terminus that contains multiple SH3 domain-binding motifs. We showed the following: 1) that both Xp95 isolated from Xenopus oocytes and a synthetic peptide containing the Src phosphorylation consensus sequence of Xp95 were phosphorylated in vitro by Src kinase and to a lesser extent by Fyn kinase; 2) Xp95 from Xenopus oocytes or eggs was recognized by an anti-phosphotyrosine antibody, and the relative abundance of tyrosine-phosphorylated Xp95 increased during oocyte maturation; and 3) microinjection of deregulated Src mRNA into Xenopus oocytes increased the abundance of tyrosine-phosphorylated Xp95. These results suggest that Xp95 is an element in a tyrosine kinase signaling pathway that may be involved in progesterone-induced Xenopus oocyte maturation.     

Annexin VII and annexin XI are tyrosine phosphorylated in peroxovanadate-treated dogs and in platelet-derived growth factor-treated rat vascular smooth muscle cells

The intraperitoneal administration of peroxovanadate results in the rapid accumulation of many tyrosine-phosphorylated proteins in the liver and kidney of treated animals. The availability of large pools of tyrosine-phosphorylated proteins derived from normal tissues facilitates the purification and identification of previously unknown targets for cellular tyrosine kinases. Using this procedure, we have thus far identified four proteins in the liver and kidney of peroxovanadate-treated dogs. Two of these, annexin VII and annexin XI, were novel and had not been previously reported to be substrates of tyrosine kinases while the remaining two, ezrin and clathrin, have been reported to be tyrosine phosphorylated in some cell culture systems. In the present study, isolated proteins were identified both by sequence analysis and immunological methods. Annexin VII and annexin XI are present in cultured rat vascular smooth muscle cells and both were tyrosine phosphorylated in response to a physiological ligand, platelet-derived growth factor-BB (PDGF-BB). Furthermore, the extent of tyrosine phosphorylation in response to PDGF-BB was augmented by the co-addition of peroxovanadate to cell cultures. In vitro phosphorylation assays showed that PDGF receptor, calcium-dependent tyrosine kinase (CADTK/Pyk-2), Src kinase, and epidermal growth factor receptor all were able to phosphorylate purified annexin VII and XI on tyrosine residues. These findings confirm the usefulness of phosphatase inhibition by peroxovanadate as a tool for identifying previously unknown physiological targets for cellular protein tyrosine kinases.     

Regulation of TRPC6 channel activity by tyrosine phosphorylation

Various hormonal stimuli and growth factors activate the mammalian canonical transient receptor potential (TRPC) channel through phospholipase C (PLC) activation. However, the precise mechanism of the regulation of TRPC channel activity remains unknown. Here, we provide the first evidence that direct tyrosine phosphorylation by Src family protein-tyrosine kinases (PTKs) is a novel mechanism for modulating TRPC6 channel activity. We found that TRPC6 is tyrosine-phosphorylated in COS-7 cells when coexpressed with Fyn, a member of the Src family PTKs. We also found that Fyn interacts with TRPC6 and that the interaction is mediated by the SH2 domain of Fyn and the N-terminal region of TRPC6 in a phosphorylation-independent manner. In addition, we demonstrated the physical association of TRPC6 with Fyn in the mammalian brain. Moreover, we showed that stimulation of the epidermal growth factor receptor induced rapid tyrosine phosphorylation of TRPC6 in COS-7 cells. This epidermal growth factor-induced tyrosine phosphorylation of TRPC6 was significantly blocked by PP2, a specific inhibitor of Src family PTKs, and by a dominant negative form of Fyn, suggesting that the direct phosphorylation of TRPC6 by Src family PTKs could be caused by physiological stimulation. Furthermore, using single channel recording, we showed that Fyn modulates TRPC6 channel activity via tyrosine phosphorylation. Thus, our findings demonstrated that tyrosine phosphorylation by Src family PTKs is a novel regulatory mechanism of TRPC6 channel activity.     

Mitochondrial processing peptidase: multiple-site recognition of precursor proteins

The Csk Homologous Kinase (CHK) has been shown to have an enzymatic activity similar to the tyrosine kinase Csk in that it down-regulates Src family kinase activity by causing phosphorylation of the Src C-terminal tyrosine residue. In megakaryocytic Mo7e cells, CHK associates with a specific phosphotyrosine juxtamembrane sequence of the SCF/KL-activated c-Kit receptor. Here, we show that in Mo7e cells, the major Src family kinase activity is p53/56(Lyn). Studies using immobilized c-Kit phosphopeptides show that Lyn is able to specifically associate with the tyrosine-phosphorylated juxtamembrane 568Y*VY*IDPT sequence of c-Kit which has previously been shown to associate with CHK. In cells over-expressing CHK by means of a recombinant vaccinia virus, we observed an elimination of the SCF/KL-stimulated Lyn kinase peak of activity observed at 2-5 minutes in cells infected with the helper T7-expressing vaccinia virus by itself. Examination of total tyrosine phosphorylation by Western blotting showed that over-expression of CHK resulted in a reduction in the levels of tyrosine phosphorylations in the range of 50-60 kDa, but had no apparent effect on c-Kit autophosphorylation. Taken together, these findings show that CHK is able to down-regulate SCF/KL-stimulated Lyn activity in megakaryocytes.     

Anaplasma phagocytophilum AnkA is tyrosine-phosphorylated at EPIYA motifs and recruits SHP-1 during early infection

Anaplasma phagocytophilum is an intracellular pathogen that infects and survives in neutrophilic granulocytes. The A. phagocytophilum genome encodes a type four secretion system (T4SS) that may facilitate intracellular survival by translocation of virulence factors, but to date, no such factors have been identified. Because T4SS-translocated proteins of several intracellular organisms undergo tyrosine phosphorylation by host cell kinases, we investigated tyrosine phosphorylation of A. phagocytophilum proteins during infection. Within minutes after incubation of A. phagocytophilum with HL-60 cells or PMN, a 190 kDa bacterial protein, AnkA, was increasingly tyrosine-phosphorylated. A. phagocytophilum binding to host cells without entry was sufficient for AnkA tyrosine phosphorylation. An in vitro Src kinase assay demonstrated that purified AnkA expressed in Escherichia coli was phosphorylated at tyrosines located at the C-terminal portion of AnkA. Similarly, AnkA expressed in COS-7 cells underwent tyrosine phosphorylation by Src at the C-terminus. The phosphorylated tyrosines were located in EPIYA motifs that display the consensus sequence for binding to SH2 domains. Immunoprecipitation studies demonstrated AnkA binding to the host cell phosphatase SHP-1 during early infection. Phosphorylation of the EPIYA motifs and the presence of the SH2 domains were necessary for AnkA-SHP-1 interaction. We conclude that AnkA is a translocated virulence factor that is tyrosine-phosphorylated by host cell kinases upon translocation into the host cell early during infection. A. phagocytophilum may manipulate the host cell through SHP-1 recruitment.     

Identification of Tyr-397 as the primary site of tyrosine phosphorylation and pp60src association in the focal adhesion kinase, pp125FAK.

A number of cellular processes, such as proliferation, differentiation, and transformation, are regulated by cell-extracellular matrix interactions. Previous studies have identified a novel tyrosine kinase, the focal adhesion kinase p125FAK, as a component of cell adhesion plaques. p125FAK was identified as a 125-kDa tyrosine-phosphorylated protein in cells transformed by the v-src oncogene. p125FAK is an intracellular protein composed of three domains: a central domain with homology to protein tyrosine kinases, flanked by two noncatalytic domains of 400 amino acids which bear no significant homology to previously cloned proteins. p125FAK is believed to play an important regulatory role in cell adhesion because it localizes to cell adhesion plaques and because its phosphorylation on tyrosine residues is regulated by binding of cell surface integrins to the extracellular matrix. Recent studies have shown that Src, through its SH2 domain, stably associates with pp125FAK and that this association prevents dephosphorylation of pp125FAK in vitro by protein tyrosine phosphatases. In this report, we identify Tyr-397 as the primary in vivo and in vitro site of p125FAK tyrosine phosphorylation and association with Src. Substituting phenylalanine for tyrosine at position 397 significantly reduces p125FAK tyrosine phosphorylation and association with Src but does not abolish p125FAK kinase activity. In addition, p125FAK kinase is able to trans-phosphorylate Tyr-397 in vitro in a kinase-deficient p125FAK variant. Phosphorylation of Tyr-397 provides a site [Y(P)AEI] that fits the consensus sequence for the binding of Src.     

Rac1 function is required for Src-induced transformation. Evidence of a role for Tiam1 and Vav2 in Rac activation by Src

The proto-oncogene c-Src has been implicated in the development and progression of a number of human cancers including those of colon and breast. Accumulating evidence indicates that activated alleles of Src may induce cell transformation through Ras-ERK-dependent and -independent pathways. Here we show that Rac1 activity is strongly elevated in Src-transformed cells and that this small G protein is a critical component of the pathway connecting oncogenic Src with cell transformation. We further show that Vav2 and the ubiquitously expressed Rac1 guanine nucleotide exchange factor Tiam1 are phosphorylated in tyrosine residues in cells transfected with active and oncogenic Src. Moreover, phosphorylation of Tiam1 in cells treated with pervanadate, a potent inhibitor of tyrosine phosphatases, was partially inhibited by the Src inhibitor SU6656. Using truncated mutants of Tiam1, we demonstrate that multiple sites can be tyrosine-phosphorylated by Src. Furthermore, Tiam1 cooperated with Src to induce activation of Rac1 in vivo and the formation of membrane ruffles. Similarly, activation of JNK and the c-jun promoter by Src were also potently increased by Tiam1. Together, these results suggest that Vav2 and Tiam1 may act as downstream effectors of Src, thereby regulating Rac1-dependent pathways that participate in Src-induced cell transformation.