Myristylation is required for Tyr-527 dephosphorylation and activation of pp60c-src in mitosis.

The chicken proto-oncoprotein c-Src is phosphorylated by p34cdc2 during mitosis concomitant with increased c-Src tyrosine kinase activity. On the basis of indirect evidence, we previously suggested that this is caused by partial dephosphorylation at Tyr-527, the phosphorylation of which suppresses c-Src kinase activity. In support of this hypothesis, we now show that treatment of cells with a protein tyrosine phosphatase inhibitor, sodium vanadate, blocks the mitotic increase in Src kinase activity. Also, we show that an amino-terminal mutation that prevents myristylation (and membrane localization) of c-Src does not interfere with the p34cdc2-mediated phosphorylations but blocks both mitotic dephosphorylation of Tyr-527 (in kinase-defective Src) and stimulation of c-Src kinase activity. Furthermore, in unsynchronized cells, the kinase activity of nonmyristylated c-Src is suppressed by 60% relative to wild-type c-Src, presumably because of increased Tyr-527 phosphorylation. Consistent with this, the Tyr-527 dephosphorylation rate measured in cell homogenates is much higher for wild-type, myristylated c-Src than for nonmyristylated c-Src. Tyr-527 phosphatase activity was primarily associated with the nonsoluble subcellular fraction. These findings suggest that the phosphatase(s) that acts on Tyr-527 is membrane bound and indicate that membrane localization of c-Src is necessary for its mitotic activation by dephosphorylation of Tyr-527.     

Regulation of pp60c-src and its interaction with polyomavirus middle T antigen in insect cells.

High yields of soluble, biologically active pp60c-src and middle t antigen (MTAg) of polyomavirus were produced in insect cells, using a baculovirus expression system. In mammalian cells, pp60c-src undergoes a regulatory phosphorylation on Tyr-527 in vivo and is autophosphorylated on Tyr-416 in vitro. In insect cells, pp60c-src was phosphorylated primarily on Tyr-416, although Tyr-527 was detectable at a low level. A kinase-negative mutant of pp60c-src was not phosphorylated on either Tyr-527 or Tyr-416 in insect cells and thus is an excellent biochemical reagent to search for the regulatory kinase that usually phosphorylates Tyr-527 in mammalian cells. MTAg synthesized in insect cells was not phosphorylated on tyrosine residues in vivo or in vitro, suggesting that it did not associate with any endogenous tyrosine kinases. However, MTAg isolated from cells coinfected with viruses encoding both MTAg and pp60c-src was phosphorylated on tyrosine residues both in vivo and in vitro.     

Dephosphorylation or antibody binding to the carboxy terminus stimulates pp60c-src.

Phosphorylation of pp60c-src at Tyr-527, six residues from the carboxy terminus, has been implicated in regulation of the protein-tyrosine kinase activity of pp60c-src. Here we show that dephosphorylation of pp60c-src by phosphatase treatment in vitro caused a 10- to 20-fold increase in pp60c-src protein-tyrosine kinase activity. Binding of specific antibody to the region of pp60c-src which contains phosphotyrosine-527 also increased kinase activity. Each treatment increased phosphorylation of added substrates and of Tyr-416 within pp60c-src by a similar mechanism that involved altered interactions with ATP and increased catalytic rate. We suggest that the phosphorylated carboxy terminus acts as an inhibitor of the protein kinase domain of pp60c-src, unless its conformation is altered by either dephosphorylation or antibody binding. The antibody additionally stimulated the phosphorylation of forms of pp60c-src that had reduced gel mobility, much like those phosphorylated in kinase reactions containing pp60c-src activated by polyomavirus medium tumor antigen. These in vitro experiments provide models for the activation of pp60c-src in cells transformed by polyomavirus. We also show that autophosphorylation of pp60c-src at Tyr-527 occurs only to a very limited extent in vitro, even when Tyr-527 is made available for phosphorylation by treatment with phosphatase. This suggests that other protein-tyrosine kinases may normally phosphorylate Tyr-527 and regulate pp60c-src in the cell.     

Protein kinase activity of FSV (Fujinami sarcoma virus) P130gag-fps shows a strict specificity for tyrosine residues

A number of oncogenic viruses encode transforming proteins with protein kinase activities apparently specific for tyrosine residues. Recent evidence has raised questions as to the substrate specificity of these kinases in general and the physiological relevance of tyrosine phosphorylation in particular. The P130gag-fps transforming protein of Fujinami sarcoma virus (FSV) is strongly phosphorylated at 2 tyrosine residues in FSV-transformed cells of which 1 (Tyr-1073) is also the major site of P130gag-fps intermolecular autophosphorylation in vitro. We have investigated the specificity of the protein kinase activity intrinsic to FSV P130gag-fps by using site-directed mutagenesis to change the codon for Tyr-1073 to those for the other commonly phosphorylated hydroxyamino acids, serine and threonine. This approach has some advantages over the use of synthetic peptides to define protein kinase recognition sites in that the protein containing the altered target site can be expressed in intact cells. In addition it allows higher order as well as primary structure of the enzyme recognition site to be considered. Neither serine nor threonine were phosphorylated when substituted for tyrosine at position 1073 of P130gag-fps indicating a stringent specificity for tyrosine as a substrate of the P130gag-fps protein kinase autophosphorylating activity. Consistent with the suggestion that tyrosine phosphorylation is of functional significance we find that these and other FSV Tyr-1073 mutants have depressed enzymatic and oncogenic capacities.     

The carboxy terminus of pp60c-src is a regulatory domain and is involved in complex formation with the middle-T antigen of polyomavirus.

A large number of mutations were introduced into the carboxy-terminal domain of pp60c-src. The level of phosphorylation on Tyr-416 and Tyr-527, the transforming activity (as measured by focus formation on NIH 3T3 cells), kinase activity, and the ability of the mutant pp60c-src to associate with the middle-T antigen of polyomavirus were examined. The results indicate that Tyr-527 is a major carboxy-terminal element responsible for regulating pp60c-src in vivo. A good but not perfect correlation exists between lack of phosphorylation at Tyr-527 and increased phosphorylation at Tyr-416, between elevated phosphorylation on Tyr-416 and activated kinase activity, and between activated kinase activity and transforming activity. Phosphorylation of Tyr-527 was insensitive to the mutation of adjacent residues, indicating that the primary sequence only has a minor role in recognition by kinases or phosphatases which regulate it in vivo. Three mutants which have in common a modified Glu-524 residue were phosphorylated on Tyr-416 and Tyr-527 and were weakly transforming. This suggests that other mechanisms besides complete dephosphorylation of Tyr-527 can lead to increased phosphorylation of Tyr-416 and activation of the transforming activity of pp60c-src. Furthermore, the residues between Asp-518 and Pro-525 were required to form a stable complex with middle-T antigen. The proximity of these sequences to Tyr-527 suggests a model in which middle-T activates pp60c-src by binding directly to this region of the molecular and thereby preventing phosphorylation of Tyr-527. Alternatively, middle-T binding may mediate a conformational change in this region, which in turn induces an alteration in the level of phosphorylation at Tyr-527 and Tyr-416.     

Modulation of the catalytic activity of the Src family tyrosine kinase Hck by autophosphorylation at a novel site in the unique domain

Autophosphorylation is a key event in the activation of protein kinases. In this study, we demonstrate that autophosphorylation of the recombinant Src family kinase Hck leads to a 20-fold increase in its specific enzymatic activity. Hck was found to autophosphorylate readily to a stoichiometry of 1.3 mol of phosphate per mol of enzyme, indicating that the kinase autophosphorylated at more than one site. Solid phase sequencing and two-dimensional mapping of the phosphopeptide fragments derived from the autophosphorylated enzyme revealed that the kinase can undergo autophosphorylation at the following two sites: (i) Tyr-388, which is located to the consensus autophosphorylation site commonly found in the activation loop of many protein kinases, and (ii) Tyr-29, which is located in the unique domain of Hck. Hck purified from mouse bone marrow-derived macrophages could also autophosphorylate in vitro at both Tyr-388 and Tyr-29, indicating that naturally occurring Hck can also autophosphorylate at Tyr-29. Furthermore, Hck transiently expressed in human embryonic kidney 293T cells was found to be phosphorylated at Tyr-29 and Tyr-388, proving that Hck can also undergo autophosphorylation at both sites in vivo. The recombinant enzyme carrying the mutation of Tyr-388 to Phe was also able to autophosphorylate at Tyr-29, albeit at a significantly slower rate. A 2-fold increase in the specific enzymatic activity was seen with this mutant despite the stoichiometry of autophosphorylation only approaching 0.2 mol of phosphate per mol of enzyme. This indicates that autophosphorylation of Tyr-29 contributes significantly to the activation of Hck. Regulation of the catalytic activity by phosphorylation of Tyr-29 in the unique domain may represent a new mechanism of regulation of Src family tyrosine kinases.     

Phosphorylation of a Src kinase at the autophosphorylation site in the absence of Src kinase activity

Exposure of cells to oxidants increases the phosphorylation of the Src family tyrosine protein kinase Lck at Tyr-394, a conserved residue in the activation loop of the catalytic domain. Kinase-deficient Lck expressed in fibroblasts that do not express any endogenous Lck has been shown to be phosphorylated at Tyr-394 following H(2)O(2) treatment to an extent indistinguishable from that seen with wild type Lck. This finding indicates that a kinase other than Lck itself is capable of phosphorylating Tyr-394. Because fibroblasts express other Src family members, it remained to be determined whether the phosphorylation of Tyr-394 was carried out by another Src family kinase or by an unrelated tyrosine protein kinase. We examined here whether Tyr-394 in kinase-deficient Lck was phosphorylated following exposure of cells devoid of endogenous Src family kinase activity to H(2)O(2). Strikingly, treatment of such cells with H(2)O(2) led to the phosphorylation of Tyr-394 to an extent identical to that seen with wild type Lck, demonstrating that Src family kinases are not required for H(2)O(2)-induced phosphorylation of Lck. Furthermore, this efficient phosphorylation of Lck at Tyr-394 in non-lymphoid cells suggests the existence of an ubiquitous activator of Src family kinases.     

A tightly associated serine/threonine protein kinase regulates phosphoinositide 3-kinase activity.

We identified a serine/threonine protein kinase that is associated with and phosphorylates phosphoinositide 3-kinase (PtdIns 3-kinase). The serine kinase phosphorylates both the 85- and 110-kDa subunits of PtdIns 3-kinase and purifies with it from rat liver and immunoprecipitates with antibodies raised to the 85-kDa subunit. Tryptic phosphopeptide maps indicate that p85 from polyomavirus middle T-transformed cells is phosphorylated in vivo at three sites phosphorylated in vitro by the associated serine kinase. The 85-kDa subunit of PtdIns 3-kinase is phosphorylated in vitro on serine at a stoichiometry of approximately 1 mol of phosphate per mol of p85. This phosphorylation results in a three- to sevenfold decrease in PtdIns 3-kinase activity. Dephosphorylation with protein phosphatase 2A reverses the inhibition. This suggests that the association of protein phosphatase 2A with middle T antigen may function to activate PtdIns 3-kinase.     

The lamin B receptor of the inner nuclear membrane undergoes mitosis-specific phosphorylation and is a substrate for p34cdc2-type protein kinase.

The lamin B receptor (LBR) is an integral protein of the inner nuclear membrane that interacts with lamin B in vitro. If contains a 204-amino acid nucleoplasmic amino-terminal domain and a hydrophobic carboxyl-terminal domain with eight putative transmembrane segments. We found cell cycle-dependent phosphorylation of LBR using phosphoamino acid analysis and phosphopeptide mapping of in vivo 32P-labeled LBR immunoprecipitated from chicken cells in interphase and arrested in mitosis. LBR was phosphorylated only on serine residues in interphase and on serine and threonine residues in mitosis. Some serine residues phosphorylated in interphase were not phosphorylated in mitosis. To identify a threonine residue specifically phosphorylated in mitosis and the responsible protein kinase, wild-type and mutant LBR nucleoplasmic domain fusion proteins were phosphorylated in vitro by p34cdc2-type protein kinase. Comparisons of phosphopeptide maps to those of in vivo 32P-labeled mitotic LBR showed that Thr188 is likely to be phosphorylated by this enzyme during mitosis. These phosphorylation/dephosphorylation events may be responsible for some of the changes in the interaction between the nuclear lamina and the inner nuclear membrane that occur during mitosis.     

Mutation of amino acids in pp60c-src that are phosphorylated by protein kinases C and A.

The product of the c-src proto-oncogene, pp60c-src, is phosphorylated at Ser-17 by cyclic AMP-dependent protein kinase A and at Ser-12 by calcium-phospholipid-dependent protein kinase C (when stimulated by 12-O-tetradecanoyl phorbol acetate). We tested the effects of Ser----Ala and Ser----Glu mutations at these sites in pp60c-src and in pp60c-src(F527) (a mutant whose transforming activities are enhanced by Tyr-527----Phe mutation) by transfecting single-, double-, and triple-mutant src expression plasmids into NIH 3T3 cells. Tryptic phosphopeptide analyses of the mutant proteins confirmed prior biochemical identifications of the phosphorylation sites and showed that neither separate nor coordinate mutations at Ser-12 and Ser-17 affected Tyr-416, Tyr-527, or Ser-48 phosphorylation or prevented mitosis-specific phosphorylations of either pp60c-src or pp60c-src(F527). Ser-12 mutation did not affect phosphorylation of the Ser-17-containing peptide, but mutation of Ser-17 significantly increased phosphorylation at Ser-12. Specific kinase activities (both with and without in vivo 12-O-tetradecanoyl phorbol acetate treatment) and the abilities of pp60c-src and pp60c-src(F527) to induce foci, transformed morphologies, and anchorage-independent growth were unaffected by any of the serine mutations. Thus, pp60c-src transforming activity in NIH 3T3 cells is relatively insensitive to phosphorylation at these sites, but there is a suggestion that Ser-17 phosphorylation may have a subtle regulatory effect.     

Phosphorylation of casein kinase II by p34cdc2 in vitro and at mitosis.

In human epidermal carcinoma A431 cells, the beta subunit of casein kinase II is phosphorylated at an autophosphorylation site and at serine 209 which can be phosphorylated in vitro by p34cdc2 (Litchfield, D. W., Lozeman, F. J., Cicirelli, M. F., Harrylock, M., Ericsson, L. H., Piening, C. J., and Krebs, E. G. (1991) J. Biol. Chem. 266, 20380-20389). Given the importance of p34cdc2 in the regulation of cell cycle events, we were interested in examining the phosphorylation of casein kinase II during different stages of the cell cycle. In this study it is demonstrated that the extent of phosphorylation of serine 209 in the beta subunit is significantly increased relative to phosphorylation of the autophosphorylation site when chicken bursal lymphoma BK3A cells are arrested at mitosis by nocodazole treatment. This result suggests that serine 209 is a likely physiological target for p34cdc2. In addition, the alpha subunit of casein kinase II also undergoes dramatic phosphorylation with an associated alteration in its electrophoretic mobility when BK3A cells or human Jurkat cells are arrested with nocodazole. Phosphopeptide mapping studies indicate that p34cdc2 can phosphorylate in vitro the same peptides on the alpha subunit that are phosphorylated in cells arrested at mitosis. These phosphorylation sites were localized to serine and threonine residues in the carboxyl-terminal domain of alpha. Taken together, the results of this study indicate that casein kinase II is a probable physiological substrate for p34cdc2 and suggest that its functional properties could be affected in a cell cycle-dependent manner.     

Substitution of Ser-17 of pp60c-src: biological and biochemical characterization in chicken embryo fibroblasts.

pp60c-src is phosphorylated mainly on Ser-17 and Tyr-527 in vivo. In this study, we examined the effect of the phosphorylation of Ser-17 on the properties of pp60c-src by introducing Rous sarcoma virus variants carrying pp60c-src in which Ser-17 had been substituted, into chicken embryo fibroblasts. The Ala-17 substitution in wild-type pp60c-src and pp60c-src carrying Phe-527 caused a two- to threefold elevation in the kinase activity in vitro of these proteins; the former variant resulted in no morphological changes of infected cells, whereas the latter variant transformed chicken embryo fibroblasts. Since the substitution of Tyr-527 per se has been reported to activate pp60c-src, these results suggest that the abolishment of the phosphorylation of Ser-17 does not affect noticeably the properties of pp60c-src in chicken embryo fibroblasts.     

Molecular features of the viral and cellular Src kinases involved in interactions with the GTPase-activating protein.

GTPase-activating protein (GAP) enhances the rate of GTP hydrolysis by cellular Ras proteins and is implicated in mitogenic signal transduction. GAP is phosphorylated on tyrosine in cells transformed by Rous sarcoma virus and serves as an in vitro substrate of the viral Src (v-Src) kinase. Our previous studies showed that GAP complexes stably with normal cellular Src (c-Src), although its association with v-Src is less stable. To further investigate the molecular basis for interactions between GAP and the Src kinases, we examined GAP association with and phosphorylation by a series of c-Src and v-Src mutants. Analysis of GAP association with c-Src/v-Src chimeric proteins demonstrates that GAP associates stably with Src proteins possessing low kinase activity and poorly with activated Src kinases, especially those that lack the carboxy-terminal segment of c-Src containing the regulatory amino acid Tyr-527. Phosphorylated Tyr-527 is a major determinant of c-Src association with GAP, as demonstrated by c-Src point mutants in which Tyr-527 is changed to Phe. While the isolated amino-terminal half of the c-Src protein is insufficient for stable GAP association, analysis of point substitutions of highly conserved amino acid residues in the c-Src SH2 region indicate that this region also influences Src-GAP complex formation. Therefore, our results suggest that both Tyr-527 phosphorylation and the SH2 region contribute to stable association of c-Src with GAP. Analysis of in vivo phosphorylation of GAP by v-Src mutants containing deletions encompassing the SH2, SH3, and unique regions suggests that the kinase domain of v-Src contains sufficient substrate specificity for GAP phosphorylation. Even though tyrosine phosphorylation of GAP correlates to certain extent with the transforming ability of various c-Src and v-Src mutants, our data suggest that other GAP-associated proteins may also have roles in Src-mediated oncogenic transformation. These findings provide additional evidence for the specificity of Src interactions with GAP and support the hypothesis that these interactions contribute to the biological functions of the Scr kinases.     

pp60c-src variants containing lesions that affect phosphorylation at tyrosines 416 and 527.

The biological and biochemical properties of pp60c-src are regulated, in part, by phosphorylation at Tyr-416 and Tyr-527. The tyrosine kinase and transforming activities of pp60c-src are suppressed by phosphorylation at Tyr-527, whereas full activation of pp60c-src requires phosphorylation at Tyr-416. To test specifically the significance of the negatively charged phosphate moieties on these tyrosine residues, we have substituted the codons for both residues with codons for either Glu or Gln. A negatively charged Glu at position 527 was unable to mimic a phosphorylated Tyr at this position, and, in consequence, the mutated pp60c-src was activated and transforming. Similarly, substitution of Tyr-416 with Glu was unable to stimulate the activities of the enzyme. However, mutagenesis of Tyr-416 to Gln (to form the mutant 416Q) activated the kinase activity approximately twofold over that observed for wild-type pp60c-src. When introduced into the mutant 527F (containing Phe-527 instead of Tyr), the double mutant 416Q-527F exhibited weak transforming activity. This is in contrast to the other double mutants 416E-527F and 416F-527F, which were nontransforming. The biochemical basis by which 416Q activates pp60c-src is not understood but probably involves some local conformational perturbation. Deletion of residues 519 to 524 (RH5), a region previously shown to be necessary for association with middle-T antigen, led to loss of phosphorylation at Tyr-527 and activation of the enzymatic and focus-forming activities of pp60c-src. Hence, the sequences necessary for complex formation with middle-T antigen may also be required by the kinase(s) which phosphorylates Tyr-527 in vivo. This suggests that normal cells contain cellular proteins which are analogous to middle-T antigen and whose action regulates the activity of pp60c-src by controlling phosphorylation or dephosphorylation at residue 527.     

Phosphorylation of distinct regions of f1 histone. Relationship to the cell cycle.

The phosphorylation of different amino acids in distinct regions of f1 histone was studied in highly synchronized Chinese hamster cell populations (line CHO). The purified, 32P-labeled f1 histone was bisected into NH2-terminal and COOH-terminal fragments with N-bromosuccinimide. Tryptic phosphopeptides from these fragments were resolved using sequential high voltage electrophoretic steps on paper. No phosphorylation was observed in early G1-arrested cells. Interphase phosphorylation began in late G1 in the COOH-terminal portion of the molecule on serine. This event continued throughout S phase and persisted into mitosis. However, in mitosis additional phosphorylation was observed in the COOH-terminal portion of the molecule on threonine, and for the only time in the CHO cell cycle the NH2-terminal portion of the molecule was also phosphorylated on both serine and threonine. The peptide studies thus predicted that a minimum of four sites (two serine and two threonine) were phosphorylated in the f1 histone of mitotic CHO cells. This was confirmed using electrophoresis in long polyacrylamide gels.     

Phosphorylation of mixed lineage kinase MLK3 by cyclin-dependent kinases CDK1 and CDK2 controls ovarian cancer cell division

Mixed lineage kinase 3 (MLK3) is a serine/threonine mitogen-activated protein kinase kinase kinase that promotes the activation of multiple mitogen-activated protein kinase pathways and is required for invasion and proliferation of ovarian cancer cells. Inhibition of MLK activity causes G2/M arrest in HeLa cells; however, the regulation of MLK3 during ovarian cancer cell cycle progression is not known. Here, we found that MLK3 is phosphorylated in mitosis and that inhibition of cyclin-dependent kinase 1 (CDK1) prevented MLK3 phosphorylation. In addition, we observed that c-Jun N-terminal kinase, a downstream target of MLK3 and a direct target of MKK4 (SEK1), was activated in G2 phase when CDK2 activity is increased and then inactivated at the beginning of mitosis concurrent with the increase in CDK1 and MLK3 phosphorylation. Using in vitro kinase assays and phosphomutants, we determined that CDK1 phosphorylates MLK3 on Ser548 and decreases MLK3 activity during mitosis, whereas CDK2 phosphorylates MLK3 on Ser770 and increases MLK3 activity during G1/S and G2 phases. We also found that MLK3 inhibition causes a reduction in cell proliferation and a cell cycle arrest in ovarian cancer cells, suggesting that MLK3 is required for ovarian cancer cell cycle progression. Taken together, our results suggest that phosphorylation of MLK3 by CDK1 and CDK2 is important for the regulation of MLK3 and c-Jun N-terminal kinase activities during G1/S, G2, and M phases in ovarian cancer cell division.     

Mitotic activation of protein-tyrosine phosphatase alpha and regulation of its Src-mediated transforming activity by its sites of protein kinase C phosphorylation

During mitosis, the catalytic activity of protein-tyrosine phosphatase (PTP) alpha is enhanced, and its inhibitory binding to Grb2, which specifically blocks Src dephosphorylation, is decreased. These effects act synergistically to activate Src in mitosis. We show here that these effects are abrogated by mutation of Ser180 and/or Ser204, the sites of protein kinase C-mediated phosphorylation within PTPalpha. Moreover, either a Ser-to-Ala substitution or serine dephosphorylation specifically eliminated the ability of PTPalpha to dephosphorylate and activate Src even during interphase. This explains why the substitutions eliminated PTPalpha transforming activity, even though PTPalpha interphase dephosphorylation of nonspecific substrates was only slightly decreased. This occurred without change in the phosphorylation of PTPalpha at Tyr789, which is required for "phosphotyrosine displacement" during Src dephosphorylation. Thus, in addition to increasing PTPalpha nonspecific catalytic activity, Ser180 and Ser204 phosphorylation (along with Tyr789 phosphorylation) regulates PTPalpha substrate specificity. This involves serine phosphorylation-dependent differential modulation of the affinity of Tyr(P)789 for the Src and Grb2 SH2 domains. The results suggest that protein kinase C may participate in the mitotic activation of PTPalpha and Src and that there are intramolecular interactions between the PTPalpha C-terminal and membrane-proximal regions that are regulated, at least in part, by serine phosphorylation.     

Src kinase mediates the regulation of phospholipase C-gamma activity by glycosphingolipids

Glucosylceramide-based glycosphingolipids have been previously demonstrated to regulate negatively the formation of inositol 1,4,5-trisphosphate by phospholipase C-gamma1. In the present study, the depletion of endogenous glucosylceramide by D-t-EtDO-P4 in cultured ECV304 cells induced autophosphorylation of Src kinase at tyrosine residue 418 within the catalytic loop and dephosphorylation of Src kinase at tyrosine residues 529 within the carboxyl-terminal regulatory region. Phosphotransferase activities of Src kinase were also induced in the glucosylceramide-depleted cells. c-Src kinase activity and phosphorylations at Src Tyr-418 and epidermal growth factor (EGF) receptor Tyr-1068 were significantly enhanced by bradykinin in response to 100 nm D-t-EtDO-P4 compared with control cells. The phosphorylation and dephosphorylation on Tyr-418 and Tyr-529 residues of c-Src were reversed by treatment of 4-amino-5-(4-chlorophenyl)-7-t-butyl(pyrazolo)[3,4-d]pyrimidine (PP2), an inhibitor of Src kinase, in control cells. Glucosylceramide-depleted cells resisted treatment with PP2, and both phosphorylation of Tyr-418 and dephosphorylation of Tyr-529 induced by depletion of glucosylceramide were maintained. Compared with untreated cells, tyrosine phosphorylation of phospholipase C-gamma1 was enhanced by EGF stimulation in glucosylceramide-depleted cells, associated with enhanced tyrosine phosphorylation of the EGF receptor at Tyr-1068 and Tyr-1086 stimulated by EGF. The Src inhibitor, PP2, significantly blocked EGF-induced tyrosine phosphorylation of phospholipase C-gamma1 in control cells, whereas in glucosylceramide-depleted cells, suppression of Src kinase activity by PP2 toward EGF-induced tyrosine phosphorylation of phospholipase C-gamma1 was less significant. Thus the activation of Src kinase by depletion of glucosylceramide-based glycosphingolipids in cultured ECV304 cells is a critical up-stream event in the activation of phospholipase C-gamma1.     

Src family kinases are required for integrin-mediated but not for G protein-coupled receptor stimulation of focal adhesion kinase autophosphorylation at Tyr-397

Plating suspended Swiss 3T3 cells onto fibronectin-coated dishes promoted phosphorylation of endogenous focal adhesion kinase (FAK) at Tyr-397, the major autophosphorylation site, and at Tyr-577, located in the activation loop, as revealed by site-specific antibodies that recognize the phosphorylated form of these residues. Treatment with the selective Src family kinase inhibitor pyrazolopyrimidine 2 (PP-2) markedly reduced the phosphorylation of both Tyr-397 and Tyr-577 induced by fibronectin. Furthermore, fibronectin-mediated FAK phosphorylation at Tyr-397 was dramatically reduced in SYF cells (deficient in Src, Yes, and Fyn expression). Stimulation of Swiss 3T3 cells with bombesin also induced a rapid increase in the phosphorylation of endogenous FAK at Tyr-397. In contrast to the results obtained with fibronectin, PP-2 did not prevent FAK Tyr-397 phosphorylation stimulated by bombesin at a concentration (10 micrometer) that suppressed bombesin-induced FAK Tyr-577 phosphorylation. Similarly, PP-2 did not prevent Tyr-397 phosphorylation in Swiss 3T3 cells stimulated with other G protein-coupled receptor agonists including vasopressin, bradykinin, endothelin, and lysophosphatidic acid. Lysophosphatidic acid also induced FAK phosphorylation at Tyr-397 in SYF cells. Our results identify, for first time, the existence of Src-dependent and Src-independent pathways leading to FAK autophosphorylation at Tyr-397 stimulated by adhesion-dependent signals and G protein-coupled receptor agonists in the same cell.     

Src signaling regulates completion of abscission in cytokinesis through ERK/MAPK activation at the midbody

Src family non-receptor-type tyrosine kinases regulate a wide variety of cellular events including cell cycle progression in G(2)/M phase. Here, we show that Src signaling regulates the terminal step in cytokinesis called abscission in HeLa cells. Abscission failure with an unusually elongated intercellular bridge containing the midbody is induced by treatment with the chemical Src inhibitors PP2 and SU6656 or expression of membrane-anchored Csk chimeras. By anti-phosphotyrosine immunofluorescence and live cell imaging, completion of abscission requires Src-mediated tyrosine phosphorylation during early stages of mitosis (before cleavage furrow formation), which is subsequently delivered to the midbody through Rab11-driven vesicle transport. Treatment with U0126, a MEK inhibitor, decreases tyrosine phosphorylation levels at the midbody, leading to abscission failure. Activated ERK by MEK-catalyzed dual phosphorylation on threonine and tyrosine residues in the TEY sequence, which is strongly detected by anti-phosphotyrosine antibody, is transported to the midbody in a Rab11-dependent manner. Src kinase activity during the early mitosis mediates ERK activation in late cytokinesis, indicating that Src-mediated signaling for abscission is spatially and temporally transmitted. Thus, these results suggest that recruitment of activated ERK, which is phosphorylated by MEK downstream of Src kinases, to the midbody plays an important role in completion of abscission.