Phospholipase C-gamma1 is required for survival in heat stress: involvement of protein kinase C-dependent Bcl-2 phosphorylation

The consequences of heat-induced phospholipase C-gamma1 (PLC-gamma1) phosphorylation are not known. We investigated the role of PLC-gamma1 activation and its downstream targets during the cellular response to heat stress using mouse embryonic fibroblasts genetically deficient in PLC-gamma1 (Plcg1 null MEF) and its wild type (wt MEF) as models. Treatment of wt MEF with heat resulted in temperature- and heating duration-dependent tyrosine phosphorylation of PLC-gamma1. HSP70 synthesis and the activation of extracellular signal-regulated kinases 1/2 (ERK1/2) and c-Jun N-terminal protein kinase (JNK) increased equally following heat treatment in both cell lines. However, heat-induced protein kinase C (PKC) activation was dramatically reduced in Plcg1 null MEF compared with wt MEF. Importantly, the mitochondrial localization of PKCalpha, PKC-dependent phosphorylation of Bcl-2, and cell viability in Plcg1 null MEF following heat treatment, were significantly decreased compared with the wild type. Furthermore, pretreatment with bryostatin-1, a PKC activator, enhanced Bcl-2 phosphorylation and cellular resistance to heat-induced apoptosis in Plcg1 null MEF. Taken together, these results suggest that PLC-gamma1 activation enhances cell survival through the PKC-dependent phosphorylation of Bcl-2 during the cellular response to heat stress.     

Inhibition of phospholipase C-gamma 1 augments the decrease in cardiomyocyte viability by H2O2

The present study was conducted to examine the role of a major cardiac phospholipase C (PLC) isozyme, PLC-gamma 1, in cardiomyocytes during oxidative stress. Left ventricular cardiomyocytes were isolated by collagenase digestion from adult male Sprague-Dawley rats (250-300 g) and treated with 20, 50, and 100 microM H2O2 for 15 min. A concentration-dependent (up to 50 microM) increase in the mRNA level and membrane protein content of PLC-gamma 1 was observed with H2O2 treatment. Furthermore, PLC-gamma 1 was activated in response to H2O2, as revealed by an increase in the phosphorylation of its tyrosine residues. There was a marked increase in the phosphorylation of the antiapoptotic protein Bcl-2 by H2O2; this change was attenuated by a PLC inhibitor, U-73122. Although both protein kinase C (PKC)-delta and -epsilon protein contents were increased in the cardiomyocyte membrane fraction in response to H2O2, PKC-epsilon activation, unlike PKC-delta, was attenuated by U-73122 (2 microM). Inhibition of PKC-epsilon with inhibitory peptide (0.1 microM) prevented Bcl-2 phosphorylation. Moreover, different concentrations (0.05, 0.1, and 0.2 microM) of this peptide augmented the decrease in cardiomyocyte viability in response to H2O2. In addition, a decrease in cardiomyocyte viability, as assessed by trypan blue exclusion, due to H2O2 was also seen when cells were pretreated with U-73122 and was as a result of increased apoptosis. It is therefore suggested that PLC-gamma 1 may play a role in cardiomyocyte survival during oxidative stress via PKC-epsilon and phosphorylation of Bcl-2.     

Platelet-derived growth factor increases the in vivo activity of phospholipase C-gamma 1 and phospholipase C-gamma 2.

Upon binding to its cell surface receptor, platelet-derived growth factor (PDGF) causes the tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1) and stimulates the production of diacylglycerol and inositol 1,4,5-triphosphate. We showed that following stimulation by PDGF, rat-2 cells overexpressing PLC-gamma 1 display an increase in the levels of both tyrosine-phosphorylated PLC-gamma 1 and inositol phosphates compared with the parental rat-2 cells. This increased responsiveness to PDGF is a direct effect of PLC-gamma 1 overexpression, as a cell line expressing similar levels of an enzymatically inactive point mutant of PLC-gamma 1, PLC-gamma 1 335Q, did not show elevated inositol phosphate production in response to PDGF. Hematopoietic cells express PLC-gamma 2, a PLC isoform that is closely related to PLC-gamma 1. When rat-2 cells overexpressing PLC-gamma 2 were treated with PDGF, an increase in both the tyrosine phosphorylation and the in vivo activity of PLC-gamma 2 was observed. Aluminum fluoride (AIF4-), a universal activator of PLC linked to G-proteins, did not produce an increase in the levels of inositol phosphates in either of the overexpressing cell lines compared with parental rat-2 cells, demonstrating that PLC-gamma isoforms respond specifically to a receptor with tyrosine kinase activity.     

PDGF stimulation of inositol phospholipid hydrolysis requires PLC-gamma 1 phosphorylation on tyrosine residues 783 and 1254.

PDGF binding to its receptor promotes the association with and stimulates the phosphorylation of PLC-gamma 1 at tyrosine and serine residues. Also, PDGF induces an increase in the hydrolysis of inositol phospholipids by PLC. How PDGF activates PLC was investigated by substituting phenylalanine for tyrosine at PLC-gamma 1 phosphorylation sites 771, 783, and 1254 and expressing the mutant enzymes in NIH 3T3 cells. Phenylalanine substitution at Tyr-783 completely blocked the activation of PLC by PDGF, whereas mutation at Try-1254 inhibited and mutation at Tyr-771 enhanced the response. Like the wild type, PLC-gamma 1 substituted with phenylalanine at Tyr-783 became associated with the PDGF receptor and underwent phosphorylation at serine residues in response to PDGF. These results suggest that PLC-gamma 1 is the PLC isozyme that mediates PDGF-induced inositol phospholipid hydrolysis, that phosphorylation on Tyr-783 is essential for PLC-gamma 1 activation. These results provide direct evidence that growth factor receptors activate the function of intracellular protein by tyrosine phosphorylation.     

Mathematical model of phosphatidylinositol-4,5-bisphosphate hydrolysis mediated by epidermal growth factor receptor generating diacylglycerol

Phosphatidylinositol-4,5-bisphosphate (PIP2) is hydrolyzed in response to the tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) and plays an important role in regulating cell proliferation and differentiation through the generation of second messengers diacylglycerol (DAG) and trisphosphate inositol (IP3) which lead to the activation of protein kinase C (PKC) and increased levels of intracellular calcium, respectively. In the paper, a mathematical model was established to simulate the accumulation of DAG due to PIP2 hydrolysis mediated by EGFR. Molecular mechanisms between DAG, PIP2, EGFR and phosphatidylinositol transfer protein (PITP) were explained successfully, and positive cooperativity which existed between phospholipase C-gamma1 (PLC-gamma1) and PIP2 was also explained. In the model the effects of parameters on simulation of PIP2 hydrolysis were analyzed and the efficacies of some molecular intervention strategies were predicted. To test the coherence between the model and the biological response to epidermal growth factor (EGF) in cells, the levels of DAG and the tyrosine phosphorylation-EGFRs in NIH3T3 mouse embryonic fibroblast (MEF) were determined by biochemical experiments which showed that the accumulation of DAG was a sigmoidal function of phosphorylation-EGFR concentration, and the consistency between the mathematical model and experimental results was confirmed. In brief, this mathematical model provided a new idea for the further study of the dynamic change of biological characteristics in inositol phospholipid hydrolysis, predicting the efficacy of molecular intervention and the relationship between the metabolisms of inositol phospholipid and other signal transduction pathways.     

Tyrosine phosphorylation of phospholipase C-gamma 2 upon cross-linking of membrane Ig on murine B lymphocytes.

When membrane Ig (mIg) on the surface of B lymphocytes is cross-linked using anti-Ig antibodies, the enzyme phospholipase C (PLC) is activated to cleave inositol phospholipids. Tyrosine kinase inhibitors have been reported to inhibit this event. Therefore, we investigated the effect of cross-linking of mIg on the state of tyrosine phosphorylation of PLC activity in two murine B cell lines and in normal resting mouse B cells. Proteins from lysates of stimulated or unstimulated cells were immunoprecipitated with an antiphosphotyrosine antibody and subsequently assayed for PLC activity. Treatment of the B cell line WEHI-231 with anti-IgM led within 15 to 30 s to a 10- to 20-fold increase in tyrosine-phosphorylated PLC activity. Inositol trisphosphate generation by WEHI-231 cells stimulated under the same conditions demonstrated similar kinetics. Normal resting B cells treated with anti-IgM or anti-IgD demonstrated 2.5- and 4-fold increases, respectively, of tyrosine-phosphorylated PLC activity. To identify the isozyme of PLC that was phosphorylated, we immunoprecipitated PLC-gamma 1 or PLC-gamma 2 with specific antibodies and assessed the amount of tyrosine phosphorylation of these proteins by antiphosphotyrosine immunoblotting. Treatment of WEHI-231 or Bal17 cells with anti-IgM induced an increase in PLC-gamma 2 tyrosine phosphorylation over background levels. There was no detectable tyrosine phosphorylation of PLC-gamma 1 in treated or untreated WEHI-231 cells, whereas anti-IgM-treated Bal17 cells did exhibit low but detectable levels of tyrosine phosphorylation of PLC-gamma 1. In normal resting mouse B cells, there was no detectable PLC-gamma 1, but PLC-gamma 2 was abundant. These observations suggest that PLC-gamma 2 is a significant substrate for the mIg-activated protein tyrosine kinase and may be responsible for mediating mIg stimulation of inositol phospholipid hydrolysis in murine B cells.     

Activation of vascular endothelial growth factor receptor-3 and its downstream signaling promote cell survival under oxidative stress

Reactive oxygen species (ROS) mediate cell damage and have been implicated in the pathogenesis of diseases that involve endothelial injury. Cells possess antioxidant systems, including intracellular antioxidants and ROS scavenging enzymes, that control the redox state and prevent cell damage. In addition to intracellular antioxidants, certain growth factor receptors can be activated under oxidative stress and trigger downstream cell survival signaling cascades. Vascular endothelial growth factor receptor-3 (VEGFR-3) is a primary modulator of lymphatic endothelial proliferation and survival. Here, we provide evidence that activation of VEGFR-3 signaling in response to hydrogen peroxide (H(2)O(2)) promotes endothelial cell survival. Treatment with H(2)O(2) induced the tyrosine phosphorylation of VEGFR-3 and its association with the signaling adaptor proteins Shc, growth factor receptor binding protein 2, Sos, p85, SHP-2, and phospholipase C-gamma. Of note, a hereditary lymphoedema-linked mutant of VEGFR-3 was not phosphorylated by H(2)O(2) treatment. Isoforms of protein kinase C (PKC), alpha and delta, were also tyrosine-phosphorylated after H(2)O(2) stimulation. However, only the delta isoform of PKC was required for H(2)O(2)-induced phosphorylation of VEGFR-3. The tyrosine phosphorylation of VEGFR-3 or isoforms of PKC was completely inhibited by treatment with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, a specific inhibitor for Src family kinases, indicating that Src family kinases are upstream of PKC and VEGFR-3. Furthermore, expression of the wild-type but not the lymphoedema-linked mutant form of VEGFR-3 in porcine artery endothelial cells significantly enhanced the activation of Akt after H(2)O(2) stimulation. Consistent with these biochemical changes, we observed that expression and activation of the wild-type but not the mutant form of VEGFR-3 inhibited H(2)O(2)-induced apoptosis. These studies suggest that VEGFR-3 protects against oxidative damage in endothelial cells, and that patients with hereditary lymphoedema may be susceptible to ROS-induced cell damage.     

Bi-directional regulation of Ser-985 phosphorylation of c-met via protein kinase C and protein phosphatase 2A involves c-Met activation and cellular responsiveness to hepatocyte growth factor

Previous studies indicated that treatment of cells with 12-O-tetradecanoylphorbol-13-acetate induced phosphorylation of Ser-985 at the juxtamembrane of c-Met, the receptor tyrosine kinase for hepatocyte growth factor (HGF), and this was associated with decreased tyrosine phosphorylation of c-Met. However, the regulatory mechanisms and the biological significance of the Ser-985 phosphorylation in c-Met remain unknown. When A549 human lung cancer cells were exposed to oxidative stress with H(2)O(2), H(2)O(2) treatment induced phosphorylation of Ser-985, but this was abrogated by an inhibitor for protein kinase C (PKC). Likewise, treatment of cells with NaF (an inhibitor of protein phosphatases) allowed for phosphorylation of Ser-985, and a protein phosphatase responsible for dephosphorylation of Ser-985 was identified to be protein phosphatase 2A (PP2A). The effects of PKC inhibitors revealed that PKCdelta and -epsilon were responsible for the Ser-985 phosphorylation of c-Met, and pull-down analysis indicated that associations of PKCdelta and -epsilon with c-Met may be involved in the regulation of Ser-985 phosphorylation of c-Met. Instead, PP2A was constitutively associated with c-Met, whereas its activity to dephosphorylate Ser-985 of c-Met was decreased when cells were exposed to H(2)O(2). Addition of HGF to A549 cells in culture induced c-Met tyrosine phosphorylation, the result being mitogenic response and cell scattering. In contrast, in the presence of H(2)O(2) stress, HGF-dependent tyrosine phosphorylation of c-Met was largely suppressed with a reciprocal relationship to Ser-985 phosphorylation, and this event was associated with abrogation of cellular responsiveness to HGF. These results indicate that Ser-985 phosphorylation of c-Met is bi-directionally regulated through PKC and PP2A, and the Ser-985 phosphorylation status may provide a unique mechanism that confers cellular responsiveness/unresponsivenss to HGF, depending on extracellular conditions.     

A site of tyrosine phosphorylation in the C terminus of the epidermal growth factor receptor is required to activate phospholipase C.

Cells expressing mutant epidermal growth factor (EGF) receptors have been used to study mechanisms through which EGF increases phospholipase C (PLC) activity. C-terminal truncation mutant EGF receptors are markedly impaired in their ability to increase inositol phosphate formation compared with wild-type EGF receptors. Mutation of the single tyrosine self-phosphorylation site at residue 992 to phenylalanine in an EGF receptor truncated at residue 1000 abolished the ability of EGF to increase inositol phosphate formation. C-terminal deletion mutant receptors that are impaired in their ability to increase inositol phosphate formation effectively phosphorylate PLC-gamma at the same tyrosine residues as do wild-type EGF receptors. EGF enhances PLC-gamma association with wild-type EGF receptors but not with mutant receptors lacking sites of tyrosine phosphorylation. These results indicate that formation of a complex between self-phosphorylated EGF receptors and PLC-gamma is necessary for enzyme activation in vivo. We propose that both binding of PLC-gamma to activated EGF receptors and tyrosine phosphorylation of the enzyme are necessary to elicit biological responses. Kinase-active EGF receptors lacking sites of tyrosine phosphorylation are unable to signal increased inositol phosphate formation and increases in cytosolic Ca2+ concentration.     

Insulin-like growth factor type-1 receptor transactivation in vasoactive peptide and oxidant-induced signaling pathways in vascular smooth muscle cells

Transactivation of epidermal growth factor receptor (EGFR) is a well-documented mechanism by which vasoactive peptides and H2O2 elicit their cellular responses. However, a role for the insulin-like growth factor type-1 receptor (IGF-1R) transactivation in mediating the effects of angiotensin II (Ang II) and H2O2 in vascular smooth muscle cells from different artery types have also been recently recognized. By using a series of pharmacological inhibitors of various growth factor receptor tyrosine kinases and a direct analysis of the phosphorylation status of the beta-subunit of IGF-1R, a requirement of this growth factor receptor in Ang II and H2O2 response has been demonstrated. This review discusses some of the studies that highlight the importance of IGF-1R transactivation in mediating Ang II- and H2O2-induced mitogen-activated protein kinase and protein kinase B signaling pathways.     

Physiological Requirement for Both SH2 Domains for Phospholipase C-γ1 Function and Interaction with Platelet-Derived Growth Factor Receptors

Two approaches have been utilized to investigate the role of individual SH2 domains in growth factor activation of phospholipase C-gamma1 (PLC-gamma1). Surface plasmon resonance analysis indicates that the individual N-SH2 and C-SH2 domains are able to specifically recognize a phosphotyrosine-containing peptide corresponding to Tyr 1021 of the platelet-derived growth factor (PDGF) beta receptor. To assess SH2 function in the context of the full-length PLC-gamma1 molecule as well as within the intact cell, PLC-gamma1 SH2 domain mutants, disabled by site-directed mutagenesis of the N-SH2 and/or C-SH2 domain(s), were expressed in Plcg1(-/-) fibroblasts. Under equilibrium incubation conditions (4 degrees C, 40 min), the N-SH2 domain, but not the C-SH2 domain, was sufficient to mediate significant PLC-gamma1 association with the activated PDGF receptor and PLC-gamma1 tyrosine phosphorylation. When both SH2 domains in PLC-gamma1 were disabled, the double mutant did not associate with activated PDGF receptors and was not tyrosine phosphorylated. However, no single SH2 mutant was able to mediate growth factor activation of Ca2+ mobilization or inositol 1,4,5-trisphosphate (IP3) formation. Subsequent kinetic experiments demonstrated that each single SH2 domain mutant was significantly impaired in its capacity to mediate rapid association with activated PDGF receptors and become tyrosine phosphorylated. Hence, when assayed under physiological conditions necessary to achieve a rapid biological response (Ca2+ mobilization and IP3 formation), both SH2 domains of PLC-gamma1 are essential to growth factor responsiveness.     

Oxidative stress induces protein kinase D activation in intact cells. Involvement of Src and dependence on protein kinase C

Protein kinase D (PKD) is a protein serine kinase that is directly stimulated in vitro by phorbol esters and diacylglycerol in the presence of phospholipids, and activated by phorbol esters, neuropeptides, and platelet-derived growth factor via protein kinase C (PKC) in intact cells. Recently, oxidative stress was shown to activate transfected PKC isoforms via tyrosine phosphorylation, but PKD activation was not demonstrated. Here, we report that oxidative stress initiated by addition of H(2)O(2) (0.15-10 mm) to quiescent Swiss 3T3 fibroblasts activates PKD in a dose- and time- dependent manner, as measured by autophosphorylation and phosphorylation of an exogenous substrate, syntide-2. Oxidative stress also activated transfected PKD in COS-7 cells but not a kinase-deficient mutant PKD form or a PKD mutant with critical activating serine residues 744 and 748 mutated to alanines. Genistein, or the specific Src inhibitors PP-1 and PP-2 (1-10 micrometer) inhibited H(2)O(2)-mediated PKD activation by 45%, indicating that Src contributes to this signaling pathway. PKD activation by H(2)O(2) was also selectively potentiated by cotransfection of PKD together with an active form of Src (v-Src) in COS-7 cells, as compared with PDB-mediated activation. The specific phospholipase C inhibitor, partly blocked H(2)O(2)-mediated but not PDB-mediated PKD activation. In contrast, PKC inhibitors blocked H(2)O(2) or PDB-mediated PKD activation essentially completely, suggesting that whereas Src mediates part of its effects via phospholipase C activation, PKC acts more proximally as an upstream activator of PKD. Together, these studies reveal that oxidative stress activates PKD by initiating distinct Src-dependent and -independent pathways involving PKC.     

Activation of insulin-like growth factor type-1 receptor is required for H2O2-induced PKB phosphorylation in vascular smooth muscle cells

Evidence accumulated in recent years has revealed a potential role for reactive oxygen species (ROS) in the pathophysiology of cardiovascular diseases. However, the precise mechanisms by which ROS contribute to the development of these diseases are not fully established. Previous work from our laboratory has indicated that exogenous hydrogen peroxide (H2O2) activates several signaling protein kinases, such as extracellular signal-regulated kinase 1 and 2 (ERK1/2) and protein kinase B (PKB) in A10 vascular smooth muscle cells (VSMC). However, the upstream elements responsible for this activation remain unclear. Although a role for epidermal growth factor receptor (EGFR) protein tyrosine kinase (PTK) in H2O2-induced ERK1/2 signaling has been suggested, the contribution of this PTK or other receptor or nonreceptor PTKs to PKB activation is not well defined in VSMC. In this study, we used pharmacological inhibitors to investigate the role of receptor and Src-family-PTKs in H2O2-induced PKB phosphorylation. AG1478, a specific inhibitor of EGFR, failed to attenuate the H2O2-induced increase in PKB Ser473 phosphorylation, whereas AG1024, an inhibitor of insulin-like growth factor type1 receptor (IGF-1R)-PTK, almost completely blocked this response. H2O2 treatment also enhanced tyrosine phosphorylation of the IGF-1Rbeta subunit, which was significantly inhibited by AG1024 pretreatment of cells. Furthermore, pharmacological inhibition of Src by PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazole(3,4-d) pyrimidine) decreased PKB phosphorylation. Moreover, H2O2-induced PKB phosphorylation was associated with increased tyrosine phosphorylation of c-Src and Pyk2 in an AG1024- and PP2-inhibitable manner. In conclusion, these data provide evidence of the contribution of IGF-1R-PTK in initiating H2O2-evoked PKB phosphorylation in A10 VSMC, with an intermediary role for c-Src and Pyk2 in this process.     

The phosphorylation of phospholipase C-gamma1, Raf-1, MEK, and ERK1/2 induced by a conserved retroviral peptide

A synthetic 17-amino acid peptide (CKS-17) homologous to a highly conserved region of human and animal retroviral transmembrane proteins has been found to exhibit suppressive properties for numerous immune functions. It has been shown that CKS-17 causes an imbalance of human types 1 and 2 cytokines and inhibition of the immune responses of lymphocytes, monocytes, and macrophages. CKS-17 induced increased intracellular levels of cAMP, which plays an important role in regulation of cytokine biosynthesis. In this study, using a Jurkat T-cell line and Western blot analysis, CKS-17 induced phosphorylation of PLC-gamma1, Raf-1, MEK and ERK1/2. Using a PLC selective inhibitor U73122 or PLC-gamma1-deficient Jurkat cell line, phosphorylation induced by CKS-17 of ERK1/2, PLC-gamma1, or Raf-1, respectively, were undetectable or significantly reduced. Reintroduction of PLC-gamma1 into the PLC-gamma1-deficient Jurkat cells restored the phosphorylation of ERK1/2 and PLC-gamma1 induced by CKS-17. Further, pretreatment of Jurkat cells with PKC inhibitors blocks the phosphorylation of Raf-1, MEK, and ERK1/2 induced by CKS-17. These results indicate that CKS-17 induces the PLC-gamma1-PKC-Raf-1-MEK-ERK1/2 signaling pathway.     

Inhibition of CD3-linked phospholipase C by phorbol ester and by cAMP is associated with decreased phosphotyrosine and increased phosphoserine contents of PLC-gamma 1.

The mechanisms by which phorbol 12-myristate 13-acetate (PMA) and cAMP attenuate the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) induced by ligation of the T-cell antigen receptor complex (TCR) was studied in the human Jurkat T-cell line. It has previously been shown that stimulation of Jurkat cells with antibodies to CD3, components of the TCR, elicits a rapid and transient phosphorylation of phospholipase C (PLC)-gamma 1, the predominant PLC isozyme in Jurkat cells, at multiple tyrosine residues and that such tyrosine phosphorylation leads to activation of PLC-gamma 1. Prior incubation of Jurkat cells with PMA or forskolin, which increases intracellular cAMP concentrations, prevented tyrosine phosphorylation of PLC-gamma 1 as well as the hydrolysis of PtdIns 4,5-P2 induced by ligation of CD3. Dose-response curves of PMA and of forskolin for the inhibition of PLC-gamma 1 tyrosine phosphorylation and of PtdIns 4,5-P2 hydrolysis were similar. These results suggest that the inhibition of PtdIns 4,5-P2 hydrolysis by PMA and cAMP is attributable to reduced tyrosine phosphorylation of PLC-gamma 1. Treatment of Jurkat cells with PMA or forskolin stimulated the phosphorylation of PLC-gamma 1 at serine 1248. PMA treatment also elicited the phosphorylation of PLC-gamma 1 at an unidentified serine site. Phosphopeptide map analysis indicated that the sites of PLC-gamma 1 phosphorylated in Jurkat cells treated with PMA and forskolin are the same as those phosphorylated in vitro by protein kinase C (PKC) and cAMP-dependent protein kinase (PKA), respectively. Stimulation of Jurkat cells with antibodies to CD3 also elicited phosphorylation of PLC-gamma 1 at serine 1248 and at the unidentified serine site phosphorylated in PLC-gamma 1 from PMA-treated cells. Thus, phosphorylation of PLC-gamma 1 by PKC or PKA at serine 1248 may modulate the interaction of PLC-gamma 1 with the protein tyrosine kinase or the protein tyrosine phosphatase; this altered interaction may, at least in part, be responsible for the decreased tyrosine phosphorylation of PLC-gamma 1 seen in PMA- and forskolin-treated Jurkat cells. Furthermore, in the absence of PMA, activation of PKC by diacylglycerol provides a negative feedback signal responsible for reducing the phosphotyrosine contents of PLC-gamma 1.     

Interleukin-6-induced tyrosine phosphorylation of phospholipase C-gamma1 in PC12 cells

Phospholipase C (PLC)-gamma1 plays a pivotal role in the signal transduction pathway mediated by growth factors. In this study, we found that neurite outgrowth of pheochromocytoma (PC12) cells was significantly induced by interleukin-6 (IL-6). Stimulation of PC12 cells with IL-6 led to tyrosine phosphorylation of PLC-gamma1 in a dose- and time-dependent manner. IL-6 stimulation also increased the hydrolysis of phosphatidylinositol 4,5-bisphosphate. Accumulation of total inositol phosphate as well as tyrosine phosphorylation of PLC-gamma1 was inhibited by the pretreatment of protein kinase inhibitors such as genistein and staurosporine. These results suggest that PLC-gamma1 may be involved in the signal transduction pathway of IL-6-induced PC12 cell differentiation.     

Overexpression of phospholipase C-gamma1 suppresses UVC-induced apoptosis through inhibition of c-fos accumulation and c-Jun N-terminal kinase activation in PC12 cells.

Ultraviolet-C (UVC) irradiation induces DNA damage and UVC-irradiated cells undergo cell growth arrest to repair the damaged DNA or the induction of apoptosis to prevent the risk of neoplastic transformation. Phospholipase C-gamma1 (PLC-gamma1) is a mediator of growth factor induced-signal cascade, catalyzing the hydrolysis of phosphatidyl 4,5-bisphosphate to generate second messengers, diacylglycerol and inositol 1,4,5-trisphosphate (IP(3)). PLC-gamma1 is activated by phosphorylation of tyrosine residues upon occupation of cell surface receptors by growth factors and plays an important role in controlling cellular proliferation and differentiation. In this study, we found that PLC-gamma1 was tyrosine phosphorylated within 2.5 min after UVC irradiation. To investigate the role of UVC-induced tyrosine phosphorylation of PLC-gamma1, we compared the effect of UVC between PLC-gamma1 overexpressing cells and empty vector transfected cells. Overexpression of PLC-gamma1 inhibited UVC-induced sub-diploid peak and DNA fragmentation. Northern blot analysis revealed that UVC-induced c-fos mRNA accumulation was inhibited in PLC-gamma1 overexpressing cells, while c-jun expression was not affected. In addition, UVC-induced activation of c-Jun N-terminal kinase (JNK) was significantly suppressed in PLC-gamma1 overexpressing cells. These results suggest that PLC-gamma1 may associate with the protective function against the UVC-induced cell death progression via the inhibition of accumulation of c-fos mRNA and the inhibition of JNK kinase activity.     

Stimulus-specific differences in protein kinase C delta localization and activation mechanisms in cardiomyocytes

Protein kinase C (PKC) isoforms play key roles in the regulation of cardiac contraction, ischemic preconditioning, and hypertrophy/failure. Models of PKC activation generally focus on lipid cofactor-induced PKC translocation to membranes. This study identifies tyrosine phosphorylation as an additional mechanism that regulates PKC delta actions in cardiomyocytes. Using immunoblot analysis with antibodies to total PKC delta and PKC delta-pY(311), we demonstrate that PKC delta partitions between soluble and particulate fractions (with little Tyr(311) phosphorylation) in resting cardiomyocytes. Phorbol 12-myristate 13-acetate (PMA) promotes PKC delta translocation to membranes and phosphorylation at Tyr(311). H(2)O(2) also increases PKC delta-pY(311) in association with its release from membranes. Both PMA- and H(2)O(2)-dependent increases in PKC delta-pY(311) are mediated by Src family kinases, but they occur via different mechanisms. The H(2)O(2)-dependent increase in PKC delta-pY(311) results from Src activation and increased Src-PKC delta complex formation. The PMA-dependent increase in PKC delta-pY(311) results from a lipid cofactor-induced conformational change that renders PKC delta a better substrate for phosphorylation by precomplexed Src kinases (without Src activation). PKC delta-Y(311) phosphorylation does not grossly alter the kinetics of PMA-dependent PKC delta down-regulation. Rather, tyrosine phosphorylation regulates PKC delta kinase activity. PKC delta is recovered from the soluble fraction of H(2)O(2)-treated cardiomyocytes as a tyrosine-phosphorylated, lipid-independent enzyme with altered substrate specificity. In vitro PKC delta phosphorylation by Src also increases lipid-independent kinase activity. The magnitude of this effect varies, depending upon the substrate, suggesting that tyrosine phosphorylation fine-tunes PKC delta substrate specificity. The stimulus-specific modes for PKC delta signaling identified in this study allow for distinct PKC delta-mediated phosphorylation events and responses during growth factor stimulation and oxidant stress in cardiomyocytes.     

Distinctive functions of Syk N-terminal and C-terminal SH2 domains in the signaling cascade elicited by oxidative stress in B cells

Syk plays a crucial role in the transduction of oxidative stress signaling. In this paper, we investigated the roles of Src homology 2 (SH2) domains of Syk in oxidative stress signaling, using Syk-negative DT40 cells expressing the N- or C-terminal SH2 domain mutant [mSH2(N) or mSH2(C)] of Syk. Tyrosine phosphorylation of Syk in cells expressing mSH2(N) Syk after H(2)O(2) treatment was higher than that in cells expressing wild-type Syk or mSH2(C) Syk. The tyrosine phosphorylation of wild-type Syk and mSH2(C) Syk, but not that of mSH2(N), was sensitive to PP2, a specific inhibitor of Src-family protein-tyrosine kinase. In oxidative stress, the C-terminal SH2 domain of Syk was demonstrated to be required for induction of tyrosine phosphorylation of cellular proteins, phospholipase C (PLC)-gamma2 phosphorylation, inositol 1,4, 5-triphosphate (IP(3)) generation, Ca(2)(+) release from intracellular stores, and c-Jun N-terminal kinase activation. In contrast, in mSH2(N) Syk-expressing cells, tyrosine phosphorylation of intracellular proteins including PLC-gamma2 was markedly induced in oxidative stress. The enhanced phosphorylation of mSH2(N) Syk and PLC-gamma2, however, did not link to Ca(2)(+) mobilization from intracellular pools and IP(3) generation. Thus, the N- and C-terminal SH2 domains of Syk possess distinctive functions in oxidative stress signaling.     

The Shb adaptor protein binds to tyrosine 766 in the FGFR-1 and regulates the Ras/MEK/MAPK pathway via FRS2 phosphorylation in endothelial cells

Stimulation of fibroblast growth factor receptor-1 (FGFR-1) is known to result in phosphorylation of tyrosine 766 and the recruitment and subsequent activation of phospholipase C-gamma (PLC-gamma). To assess the role of tyrosine 766 in endothelial cell function, we generated endothelial cells expressing a chimeric receptor, composed of the extracellular domain of the PDGF receptor-alpha and the intracellular domain of FGFR-1. Mutation of tyrosine 766 to phenylalanine prevented PLC-gamma activation and resulted in a reduced phosphorylation of FRS2 and reduced activation of the Ras/MEK/MAPK pathway relative to the wild-type chimeric receptor. However, FGFR-1-mediated MAPK activation was not dependent on PKC activation or intracellular calcium, both downstream mediators of PLC-gamma activation. We report that the adaptor protein Shb is also able to bind tyrosine 766 in the FGFR-1, via its SH2 domain, resulting in its subsequent phosphorylation. Overexpression of an SH2 domain mutant Shb caused a dramatic reduction in FGFR-1-mediated FRS2 phosphorylation with concomitant perturbment of the Ras/MEK/MAPK pathway. Expression of the chimeric receptor mutant and the Shb SH2 domain mutant resulted in a similar reduction in FGFR-1-mediated mitogenicity. We conclude, that Shb binds to tyrosine 766 in the FGFR-1 and regulates FGF-mediated mitogenicity via FRS2 phosphorylation and the subsequent activation of the Ras/MEK/MAPK pathway.