Phosphorylation of cyclin dependent kinase 4 on tyrosine 17 is mediated by Src family kinases

Cyclin dependent kinase 4 is a key regulator of the cell cycle and its activity is frequently deregulated in cancer. The activity of cyclin dependent kinase 4 is controlled by multiple mechanisms, including phosphorylation of tyrosine 17. This site is equivalent to tyrosine 15 of cyclin dependent kinase 1, which undergoes inhibitory phosphorylation by WEE1 and MYT1; however, the kinases that phosphorylate cyclin dependent kinase 4 on tyrosine 17 are still unknown. In the present study, we generated a phosphospecific antibody to the tyrosine 17-phosphorylated form of cyclin dependent kinase 4, and showed that this site is phosphorylated to a low level in asynchronously proliferating HCT116 cells. We purified tyrosine 17 kinases from HeLa cells and found that the Src family non-receptor tyrosine kinase C-YES contributes a large fraction of the tyrosine 17 kinase activity in HeLa lysates. C-YES also phosphorylated cyclin dependent kinase 4 when transfected into HCT116 cells, and treatment of cells with Src family kinase inhibitors blocked the tyrosine 17 phosphorylation of cyclin dependent kinase 4. Taken together, the results obtained in the present study provide the first evidence that Src family kinases, but not WEE1 or MYT1, phosphorylate cyclin dependent kinase 4 on tyrosine 17, and help to resolve how the phosphorylation of this site is regulated.     

Phosphoprotein associated with glycosphingolipid-enriched microdomains differentially modulates SRC kinase activity in brain maturation

Src family kinases (SFK) control multiple processes during brain development and function. We show here that the phosphoprotein associated with glycosphigolipid-enriched microdomains (PAG)/Csk binding protein (Cbp) modulates SFK activity in the brain. The timing and localization of PAG expression overlap with Fyn and Src, both of which we find associated to PAG. We demonstrate in newborn (P1) mice that PAG negatively regulates Src family kinases (SFK). P1 Pag1 ^-/- mouse brains show decreased recruitment of Csk into lipid rafts, reduced phosphorylation of the inhibitory tyrosines within SFKs, and an increase in SFK activity of >/ = 50%. While in brain of P1 mice, PAG and Csk are highly and ubiquitously expressed, little Csk is found in adult brain suggesting altered modes of SFK regulation. In adult brain Pag1-deficiency has no effect upon Csk-distribution or inhibitory tyrosine phosphorylation, but kinase activity is now reduced (−20–30%), pointing to the development of a compensatory mechanism that may involve PSD93. The distribution of the Csk-homologous kinase CHK is not altered. Importantly, since the activities of Fyn and Src are decreased in adult Pag1 ^-/- mice, thus presenting the reversed phenotype of P1, this provides the first in vivo evidence for a Csk-independent positive regulatory function for PAG in the brain.     

Chlamydia trachomatis tarp is phosphorylated by src family tyrosine kinases

The translocated actin recruiting phosphoprotein (Tarp) is injected into the cytosol shortly after Chlamydia trachomatis attachment to a target cell and subsequently phosphorylated by an unidentified tyrosine kinase. A role for Tarp phosphorylation in bacterial entry is unknown. In this study, recombinant C. trachomatis Tarp was employed to identify the host cell kinase(s) required for phosphorylation. Each tyrosine rich repeat of L2 Tarp harbors a sequence similar to a Src and Abl kinase consensus target. Furthermore, purified p60-src, Yes, Fyn, and Abl kinases were able to phosphorylate Tarp. Mutagenesis of potential tyrosines within a single tyrosine rich repeat peptide indicated that both Src and Abl kinases phosphorylate the same residues suggesting that C. trachomatis Tarp may serve as a substrate for multiple host cell kinases. Surprisingly, chemical inhibition of Src and Abl kinases prevented Tarp phosphorylation in culture and had no measurable effect on bacterial entry into host cells.     

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.     

Critical role for hematopoietic cell kinase (Hck)-mediated phosphorylation of Gab1 and Gab2 docking proteins in interleukin 6-induced proliferation and survival of multiple myeloma cells

Interleukin-6 (LI-6) is a known growth and survival factor in multiple myeloma via activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling cascade. In this report we show that Grb2-associated binder (Gab) family adapter proteins Gab1 and Gab2 are expressed by multiple myeloma cells; and that interleukin-6 induces their tyrosine phosphorylation and association with downstream signaling molecules. We further demonstrate that these events are Src family tyrosine kinase-dependent and specifically identify the role of hematopoietic cell kinase (Hck) as a new Gab family adapter protein kinase. Conversely, inhibition of Src family tyrosine kinases by the pyrazolopyrimidine PP2, as in kinase-inactive Hck mutants, significantly reduces IL-6-triggered activation of extracellular signal-regulated kinase and AKT-1, leading to significant reduction of multiple myeloma cell proliferation and survival. Taken together, these results delineate a key role for Hck-mediated phosphorylation of Gab1 and Gab2 docking proteins in IL-6-induced proliferation and survival of multiple myeloma cells and identify tyrosine kinases and downstream adapter proteins as potential new therapeutic targets in multiple myeloma.     

Csk enhances insulin-stimulated dephosphorylation of focal adhesion proteins.

Insulin has pleiotropic effects on the regulation of cell physiology through binding to its receptor. The wide variety of tyrosine phosphorylation motifs of insulin receptor substrate 1 (IRS-1), a substrate for the activated insulin receptor tyrosine kinase, may account for the multiple functions of insulin. Recent studies have shown that activation of the insulin receptor leads to the regulation of focal adhesion proteins, such as a dephosphorylation of focal adhesion kinase (pp125FAK). We show here that C-terminal Src kinase (Csk), which phosphorylates C-terminal tyrosine residues of Src family protein tyrosine kinases and suppresses their kinase activities, is involved in this insulin-stimulated dephosphorylation of focal adhesion proteins. We demonstrated that the overexpression of Csk enhanced and prolonged the insulin-induced dephosphorylation of pp125FAK. Another focal adhesion protein, paxillin, was also dephosphorylated upon insulin stimulation, and a kinase-negative mutant of Csk was able to inhibit the insulin-induced dephosphorylation of pp125FAK and paxillin. Although we have shown that the Csk Src homology 2 domain can bind to several tyrosine-phosphorylated proteins, including pp125FAK and paxillin, a majority of protein which bound to Csk was IRS-1 when cells were stimulated by insulin. Our data also indicated that tyrosine phosphorylation levels of IRS-1 appear to be paralleled by the dephosphorylation of the focal adhesion proteins. We therefore propose that the kinase activity of Csk, through the insulin-induced complex formation of Csk with IRS-1, is involved in insulin's regulation of the phosphorylation levels of the focal adhesion proteins, possibly through inactivation of the kinase activity of c-Src family kinases.     

Novel Role of Src in Priming Pyk2 Phosphorylation

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase (FAK) family of non-receptor tyrosine kinases and plays an important role in diverse cellular events downstream of the integrin-family of receptors, including cell migration, proliferation and survival. Here, we have identified a novel role for Src kinase in priming Pyk2 phosphorylation and subsequent activation upon cell attachment on the integrin-ligand fibronectin. By using complementary methods, we show that Src activity is indispensable for the initial Pyk2 phosphorylation on the Y402 site observed in response to cell attachment. In contrast, the initial fibronectin-induced autophosphorylation of FAK in the homologous Y397 site occurs in a Src-independent manner. We demonstrate that the SH2-domain of Src is required for Src binding to Pyk2 and for Pyk2 phosphorylation at sites Y402 and Y579. Moreover, Y402 phosphorylation is a prerequisite for the subsequent Y579 phosphorylation. While this initial phosphorylation of Pyk2 by Src is independent of Pyk2 kinase activity, subsequent autophosphorylation of Pyk2 in trans is required for full Pyk2 phosphorylation and activation. Collectively, our studies reveal a novel function of Src in priming Pyk2 (but not FAK) phosphorylation and subsequent activation downstream of integrins, and shed light on the signaling events that regulate the function of Pyk2.     

The Tyrosine Kinase Csk Dimerizes through Its SH3 Domain

The Src family kinases possess two sites of tyrosine phosphorylation that are critical to the regulation of kinase activity. Autophosphorylation on an activation loop tyrosine residue (Tyr 416 in commonly used chicken c-Src numbering) increases catalytic activity, while phosphorylation of a C-terminal tyrosine (Tyr 527 in c-Src) inhibits activity. The latter modification is achieved by the tyrosine kinase Csk (C-terminal Src Kinase), but the complete inactivation of the Src family kinases also requires the dephosphorylation of the activation loop tyrosine. The SH3 domain of Csk recruits the tyrosine phosphatase PEP, allowing for the coordinated inhibition of Src family kinase activity. We have discovered that Csk forms homodimers through interactions mediated by the SH3 domain in a manner that buries the recognition surface for SH3 ligands. The formation of this dimer would therefore block the recruitment of tyrosine phosphatases and may have important implications for the regulation of Src kinase activity.     

The SH2 domain protein Shep1 regulates the in vivo signaling function of the scaffolding protein Cas

The members of the p130Cas (Cas) family are important scaffolding proteins that orchestrate cell adhesion, migration and invasiveness downstream of integrin adhesion receptors and receptor tyrosine kinases by recruiting enzymes and structural molecules. Shep1, BCAR3/AND-34 and NSP1 define a recently identified family of SH2 domain-containing proteins that constitutively bind Cas proteins through a Cdc25-type nucleotide exchange factor-like domain. To gain insight into the functional interplay between Shep1 and Cas in vivo, we have inactivated the Shep1 gene in the mouse through Cre-mediated deletion of the exon encoding the SH2 domain. Analysis of Cas tyrosine phosphorylation in the brains of newborn mice, where Shep1 is highly expressed, revealed a strong decrease in Cas substrate domain phosphorylation in knockout compared to wild-type brains. Src family kinases bind to Cas via their SH3 and SH2 domains, which contributes to their activation, and phosphorylate multiple tyrosines in the Cas substrate domain. These tyrosine-phosphorylated motifs represent docking sites for the Crk adaptor, linking Cas to the downstream Rac1 and Rap1 GTPases to regulate cell adhesion and actin cytoskeleton organization. Accordingly, we detected lower Cas–Crk association and lower phosphorylation of the Src activation loop in Shep1 knockout brains compared to wild-type. Conversely, Shep1 transfection in COS cells increases Cas tyrosine phosphorylation. The SH2 domain is likely critical for the effects of Shep1 on Cas and Src signaling because the knockout mice express Shep1 fragments that lack the amino-terminal region including the SH2 domain, presumably due to aberrant translation from internal ATG codons. These fragments retain the ability to increase Cas levels in transfected cells, similar to full-length Shep1. However, they do not affect Cas phosphorylation on their own or in the presence of co-transfected full-length Shep1. They also do not show dominant negative effects on the activity of full-length Shep1 in vivo because the heterozygous mice, which express the fragments, have a normal life span. This is in contrast to the homozygous knockout mice, most of which die soon after birth. These data demonstrate that Shep1 plays a critical role in the in vivo regulation of Src activity and Cas downstream signaling through Crk, and suggest that the SH2 domain of Shep1 is critical for these effects.     

Introduction of p130cas signaling complex formation upon integrin-mediated cell adhesion: a role for Src family kinases.

Integrin-mediated cell adhesion triggers intracellular signaling cascades, including tyrosine phosphorylation of intracellular proteins. Among these are the focal adhesion proteins p130cas (Cas) and focal adhesion kinase (FAK). Here we identify the kinase(s) mediating integrin-induced Cas phosphorylation and characterize protein-protein interactions mediated by phosphorylated Cas. We found that expression of a constitutively active FAK in fibroblasts results in a consecutive tyrosine phosphorylation of Cas. This effect required the autophosphorylation site of FAK, which is a binding site for Src family kinases. Integrin-mediated phosphorylation of Cas was not, however, compromised in fibroblasts lacking FAK. In contrast, adhesion-induced tyrosine phosphorylation of Cas was reduced in cells lacking Src, whereas enhanced phosphorylation of Cas was observed Csk- cells, in which Src kinases are activated. These results suggest that Src kinases are responsible for the integrin-mediated tyrosine phosphorylation of Cas. FAK seems not to be necessary for phosphorylation of Cas, but when autophosphorylated, FAK may recruit Src family kinases to phosphorylate Cas. Cas was found to form complexes with Src homology 2 (SH2) domain-containing signaling molecules, such as the SH2/SH3 adapter protein Crk, following integrin-induced tyrosine phosphorylation. Guanine nucleotide exchange factors C3G and Sos were found in the Cas-Crk complex upon integrin ligand binding. These observations suggest that Cas serves as a docking protein and may transduce signals to downstream signaling pathways following integrin-mediated cell adhesion.     

Complex Formation with Focal Adhesion Kinase: A Mechanism to Regulate Activity and Subcellular Localization of Src Kinases

Tyrosine phosphorylation of focal adhesion kinase (FAK) creates a high-affinity binding site for the src homology 2 domain of the Src family of tyrosine kinases. Assembly of a complex between FAK and Src kinases may serve to regulate the subcellular localization and the enzymatic activity of members of the Src family of kinases. We show that simultaneous overexpression of FAK and pp60(c-src) or p59(fyn) results in the enhancement of the tyrosine phosphorylation of a limited number of cellular substrates, including paxillin. Under these conditions, tyrosine phosphorylation of paxillin is largely cell adhesion dependent. FAK mutants defective for Src binding or focal adhesion targeting fail to cooperate with pp60(c-src) or p59(fyn) to induce paxillin phosphorylation, whereas catalytically defective FAK mutants can direct paxillin phosphorylation. The negative regulatory site of pp60(c-src) is hypophosphorylated when in complex with FAK, and coexpression with FAK leads to a redistribution of pp60(c-src) from a diffuse cellular location to focal adhesions. A FAK mutant defective for Src binding does not effectively induce the translocation of pp60(c-src) to focal adhesions. These results suggest that association with FAK can alter the localization of Src kinases and that FAK functions to direct phosphorylation of cellular substrates by recruitment of Src kinases.     

The interaction of Src and RACK1 is enhanced by activation of protein kinase C and tyrosine phosphorylation of RACK1

RACK1 is an intracellular receptor for the serine/ threonine protein kinase C. Previously, we demonstrated that RACK1 also interacts with the Src protein-tyrosine kinase. RACK1, via its association with these protein kinases, may play a key role in signal transduction. To further characterize the Src-RACK1 interaction and to analyze mechanisms by which cross-talk occurs between the two RACK1-linked signaling kinases, we identified sites on Src and RACK1 that mediate their binding, and factors that regulate their interaction. We found that the interaction of Src and RACK1 is mediated, in part, by the SH2 domain of Src and by phosphotyrosines in the sixth WD repeat of RACK1, and is enhanced by serum or platelet-derived growth factor stimulation, protein kinase C activation, and tyrosine phosphorylation of RACK1. To the best of our knowledge, this is the first report of tyrosine phosphorylation of a member of the WD repeat family of proteins. We think that tyrosine phosphorylation of these proteins is an important mechanism of signal transduction in cells.     

Roles of cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1

Gab-1 is a multiple docking protein that is tyrosine phosphorylated by receptor tyrosine kinases such as c-Met, hepatocyte growth factor/scatter factor receptor, and epidermal growth factor receptor. We have now demonstrated that cell-cell adhesion also induces marked tyrosine phosphorylation of Gab-1 and that disruption of cell-cell adhesion results in its dephosphorylation. An anti-E-cadherin antibody decreased cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1, whereas the expression of E-cadherin specifically induced tyrosine phosphorylation of Gab-1. A relatively selective inhibitor of Src family kinases reduced cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1, whereas expression of a dominant-negative mutant of Csk increased it. Disruption of cell-cell adhesion, which reduced tyrosine phosphorylation of Gab-1, also reduced the activation of mitogen-activated protein kinase and Akt in response to cell-cell adhesion. These results indicate that E-cadherin-mediated cell-cell adhesion induces tyrosine phosphorylation by a Src family kinase of Gab-1, thereby regulating the activation of Ras/MAP kinase and phosphatidylinositol 3-kinase/Akt cascades.     

Cloning of the doding cDNA sequence of the gene for Csk tyrosine kinase from human lymphocytes

Tyrosine kinases of the Csk family play an important role in cell growth regulation and normal cell differentiation. They are also involved in carcinogenesis as oncoproteins. The main function of these tyrosine kinases is phosphorylation of tyrosine kinases of the Src family at their C-terminal regions to negatively regulate their activity. Disturbance of csk expression increases the Src tyrosine kinase activity. The full-length coding sequence of the csk cDNA was cloned from human lymphocytes. The 1624-bp cDNA consists of 12 exons and encodes a protein that has conserved SH2 and SH3 domains and is similar to human Csk tyrosine kinase by 99%. The full-length cDNA can be used to analyze the csk structure in normal or illdefined human cells.     

Obtaining a full-length encoding cDNA of Csk family tyrosine kinase from human lymphocytes

Tyrosine kinases of Csk family play important role in the cell growth regulation and normal cell differentiation and also can participate in the process of cancer genesis as oncoproteins. The main function of these tyrosine kinases is the phosphorylation of the Src family tyrosine kinases at their carboxyl terminus, which is the basis of their activity negative regulation. The disturbance of the csk gene expression leads to the increase of the Src tyrosine kinase activity. We have cloned a full-length encoding cDNA of the tyrosine kinase csk gene of human lymphocytes. 1.6-kilobase cDNA encodes the protein, which consists of 12 exons with conserved SH2 and SH3 domains. The homology between this protein and human Csk tyrosine kinase is 99%. A full-length DNA-copy of human lymphocytes RNA can be used for the analysis of csk gene structure in normal and pathologically changed human cells.