Perturbation of the platelet-derived growth factor receptor signaling by dibutyryl-camp in human astrocytoma cells

It has been shown that cAMP may perturb the polypeptide growth factor-induced nuclear events. However, the possible interactions of the cAMP-protein kinase A (cAMP-PKA) and receptor tyrosine kinase pathways in the cytosol have not been fully elucidated. In this study, we use human astrocytoma cells as a model to investigate this issue. The results show that platelet-derived growth factor (PDGF)-induced receptor autophosphorylation in human astrocytoma cells is suppressed by dibutyryl-cAMP pretreatment and such suppression is not due to changes in the ligand-receptor binding properties. Further studies show that PDGF-induced tyrosine phosphorylation of phospholipase C__γ1 (PLC__γ1) and phosphatidylinositol 3-kinase (PI 3-kinase) are also suppressed in dibutyryl-cAMP-pretreated cells. The suppression of PLC__γ1 tyrosine phosphorylation was accompanied by a decreased production of water soluble inositol phosphates. In contrast, similar treatment with normal human astrocytes potentiates the tyrosine phosphorylation of PLC__γ1 and PI 3-kinase. The results indicate that cAMP can either negatively or positively modulate the PDGF receptor tyrosine kinase activity depending on the cell types examined. © 1995 Wiley-Liss, Inc.     

Heterologous,PKC-Mediated Desensitization of Human Histamine H3 Receptors Expressed in CHO-K1 Cells

Desensitization is a major mechanism to regulate the functional response of G protein-coupled receptors. In this work we studied whether the human histamine H₃ receptor of 445 amino acids (hH₃R₄₄₅) experiences heterologous desensitization mediated by PKC activation. Bioinformatic analysis indicated the presence of Serine and Threonine residues susceptible of PKC-mediated phosphorylation on the third intracellular loop and the carboxyl terminus of the hH₃R₄₄₅. In CHO-K1 cells stably transfected with the hH₃R₄₄₅ direct PKC activation by phorbol 12-myristate 13-acetate (TPA, 200 nM) abolished H₃R-mediated inhibition of forskolin-stimulated cAMP accumulation. Activation of endogenous purinergic receptors by ATP (adenosine 5′-triphosphate, 10 μM) increased the free calcium intracellular concentration ([Ca²⁺]i) confirming their coupling to phospholipase C stimulation. Incubation with ATP also abolished H₃R-mediated inhibition of forskolin-induced cAMP accumulation, and this effect was prevented by the PKC inhibitors Ro-31-8220 and Gö-6976. Pre-incubation with TPA or ATP reduced H₃R-mediated stimulation of [³⁵S]-GTPγS binding to membranes from CHO-K1-hH₃R₄₄₅ cells by 39.7 and 54.2 %, respectively, with no change in the agonist potency, and the effect was prevented by either Ro-31-8220 or Gö-6976. Exposure to ATP or TPA also resulted in the loss of cell surface H₃Rs (−30.4 and −45.1 %) as evaluated by [³H]-NMHA binding to intact cells. These results indicate that the hH₃R₄₄₅ undergoes heterologous desensitization upon activation of receptors coupled to PKC stimulation.

     

The staurosporine analog, Ro-31-8220, induces apoptosis independently of its ability to inhibit protein kinase C

A series of bisindolylmaleimide (Bis) compounds were designed as analogs of the natural compound staurosporine (STS), which is a potent inducer of apoptosis. Many of the Bis analogs appear to be highly selective inhibitors of the protein kinase C (PKC) family, including PKC-alpha, -beta, -gamma, -delta, -epsilon, and -zeta, unlike STS, which is an inhibitor of a broad spectrum of protein kinases. In this report we describe the effects of the Bis analogs, Bis-I, Bis-II, Bis-III and Ro-31-8220 on the survival and proliferation of HL-60 cells, which have been widely used as a model cell system for studying the biological roles of PKC. Treatment of HL-60 cells with Bis-I, Bis-II, Bis-III, or Ro-31-8220 blocked phosphorylation of the PKC target protein Raf-1 with equal potency but did not appear to affect the general phosphorylation of proteins by other kinases. However, the biological effects of the Bis compounds were different: Bis-I and Bis-II had no observable effects on either cell survival or proliferation; Bis-III inhibited cell proliferation but not survival, whereas Ro-31-8220 induced apoptosis. These results indicated that the members of the PKC family which could be inhibited by the Bis analogs were required neither for survival nor proliferation of HL-60 cells. Analyses of cells treated with Ro-31-8220 showed that the apoptotic effect of Ro-31-8220 on HL-60 cells was mediated by a well-characterized transduction process of apoptotic signals: i.e., mitochondrial cytochrome c efflux and the activation of caspase-3 in the cytosol. Moreover, the ability of Ro-31-8220 to induce apoptotic activation was completely inhibited by the over-expression of the apoptotic suppressor gene, Bcl-2, in the cells. Interestingly, proliferation of the Bcl-2-over-expressing cells was still sensitive to the presence of Ro-31-8220, suggesting that the inhibitory effects of Ro-31-8220 on viability and cell proliferation were mediated by different mechanisms. In particular, the apoptotic effect of Ro-31-8220 on cells was not altered by the presence of an excess amount of the other Bis analogs, suggesting that this effect is mediated by a factor(s) other than PKC or by a mechanism which was not saturable by the other Bis analogs. Finally, structure-function analyses of compounds related to Ro-31-8220 revealed that a thioamidine prosthetic group in Ro-311-8220 was largely responsible for its apoptotic activity.     

The Src Family Kinases and Protein Kinase C Synergize to Mediate Gq-dependent Platelet Activation

The Src family kinases (SFKs) play essential roles in collagen- and von Willebrand factor (VWF)-mediated platelet activation. However, the roles of SFKs in G protein-coupled receptor-mediated platelet activation and the molecular mechanisms whereby SFKs are activated by G protein-coupled receptor stimulation are not fully understood. Here we show that the thrombin receptor protease-activated receptor 4 agonist peptide AYPGKF elicited SFK phosphorylation in P2Y₁₂ deficient platelets but stimulated minimal SFK phosphorylation in platelets lacking G_q. We have previously shown that thrombin-induced SFK phosphorylation was inhibited by the calcium chelator 5,5′-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (dimethyl-BAPTA). The calcium ionophore A23187 induced SFK phosphorylation in both wild-type and G_q deficient platelets. Together, these results indicate that SFK phosphorylation in response to thrombin receptor stimulation is downstream from G_q/Ca²⁺ signaling. Moreover, A23187-induced thromboxane A₂ synthesis, platelet aggregation, and secretion were inhibited by preincubation of platelets with a selective SFK inhibitor, PP2. AYPGKF-induced thromboxane A₂ production in wild-type and P2Y₁₂ deficient platelets was abolished by PP2, and AYPGKF-mediated P-selectin expression, integrin α_IIbβ₃ activation, and aggregation of P2Y₁₂ deficient platelets were partially inhibited by the PKC inhibitor Ro-31-8220, PP2, dimethyl-BAPTA, or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, but were abolished by Ro-31-8220 plus PP2, dimethyl-BAPTA, or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. These data indicate that Ca²⁺/SFKs/PI3K and PKC represent two alternative signaling pathways mediating G_q-dependent platelet activation.     

Differential involvement of Src family kinases in pervanadate-mediated responses in rat myometrial cells

We previously described that pervanadate, a potent tyrosine phosphatase inhibitor, induced contraction of rat myometrium via phospholipase (PL) C-gamma1 activation [Biol Reprod 54 (1996) 1383]. In this study, we found that pervanadate induced tyrosine phosphorylation of the platelet-derived growth factor (PDGF)-beta receptor, interaction of the phosphorylated PDGF receptor with the phosphorylated PLC-gamma1, production of inositol phosphates (InsPs), extracellular signal-regulated kinase (ERK) activation and DNA synthesis. All these responses were insensitive to PDGF receptor kinase inhibition or PDGF receptor down-regulation. We showed that Src family kinases were activated by pervanadate, and that InsPs production and phosphorylation of both PLC-gamma1 and the PDGF receptor were blocked by PP1, an Src inhibitor. In contrast, the stimulation of ERK by pervanadate was totally refractory to PP1. These results demonstrated that the activation of Src by pervanadate is involved in PLC-gamma1/InsPs signalling but does not play a major role in ERK activation.     

Structural and functional aspects of the receptors for platelet-derived growth factor

Platelet-derived growth factor (PDGF) is a 30 kDa dimer of disulfide-bonded A and B chains. Three isoforms of PDGF have been isolated (PDGF-AA, PDGF-AB and PDGF-BB). These bind with different affinities and specificities to two structurally related cell surface receptors, viz. the α-receptor and the β-receptor. The receptors are transmembrane proteins with an intracellular, ligand-stimulatable protein tyrosine kinase domain. Activation of the receptors is intimately associated with receptor dimerization, and available data suggest that PDGF is a divalent ligand such that one molecule of PDGF binds and dimerizes two receptor molecules. Stimulation of PDGF receptors leads to a cascade of cellular events, which have been shown to require an intact receptor tyrosine kinase activity. However, ligand-induced internalization and degradation of the β-receptor occur essentially independent of the receptor kinase activity. Receptor activation leads to the phosphorylation on tyrosine residues of three enzymes, probably by direct phosphorylation: phospholipase C-γ, phosphatidylinositol 3′ kinase and Raf-1. In certain cells, PDGF β-receptor expression is inducible such that cells in normal tissue in vivo do not express receptors; only in inflammatory lesions or when cells are explanted in vitro, are receptors being expressed. Transformation by the v-sis oncogene is mediated by an autocrine PDGF-like growth factor. Although both the α- and β-receptors are structurally related to the v-fms and v-kit oncogenes, it is not known if the PDGF receptors have a transforming potential. In conclusion, the finding of three isoforms of PDGF that interact with two structurally related receptors implies a finely tuned regulatory network, the role of which in cell growth and transformation remains to be clarified.     

On/Off-regulation of phospholipase C-γ1-mediated signal transduction

Alterations in the production and regulation of lipid second messengers can give rise to key molecular lesions that trigger tumorigenesis and cancer progression. Especially, the hydrolysis of membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), is mediated by a specific phospholipase C (PLC), which plays important roles in the regulation of various cell functions. PLC generates two intracellular messengers, diacylglycerol and inositol 1,4,5-trisphosphate, which mediate the activation of protein kinase C (PKC) and intracellular Ca²⁺ release, respectively. Among the PLC isozymes, PLC-γ1 contains two src homology (SH) 2 domains and one SH3 domain between the X and Y catalytic domains. The SH2 domains of PLC-γ1 have been implicated in the association between PLC-γ1 and activated receptor tyrosine kinases, and the SH3 domain of PLC-γ1 has been reported to be responsible for the mitogenic effect of PLC-γ1, suggesting that PLC-γ1 exerts other actions that are independent of its lipase activity and appears to be involved in the SH domains. However, the physiological role of SH domains in the regulation of PLC-γ1 is still unclear. We have recently characterized the regulation mechanism of PLC-γ1 through protein-protein interactions. We have elucidated that PLC-γ1 can serve as a guanine nucleotide exchange factor (GEF) for dynamin-1 through direct interaction via its SH3 domain. These results indicate that GEF function of PLC-γ1 for dynamin-1 may link with PLC-γ1's mitogenic actions. The SH3 domain of PLC-γ1 can mediate Sos and PIKE activation by acting as GEF, suggesting that PLC-γ1 plays an essential role on cellular proliferation through protein–protein interaction independent of its enzymatic activity. On the other hand, we have found that Cbl directly interacts with the SH3 domain of PLC-γ1 and inhibits its tyrosine phosphorylation and enzymatic activity, suggesting that PLC-γ1 can be off-regulated by protein–protein interaction. In addition, we demonstrate that Grb2 interacts with tyrosine phosphorylated PLC-γ1 and acts as an inhibitor on PLC-γ1-mediated signaling. These results suggest that Grb2, one of the key regulators of Ras/Raf/MAPK signaling pathway, may participate in the regulation of PLC-γ1. Lastly, PLC-γ1 forms a ternary complex with Jak2 and PTP-1B and negatively regulates GH-induced Jak2 phosphorylation. Taken together, our data strengthen the hypothesis that the interaction between PLC-γ1 and effector proteins plays a key role in on- or off-regulating PLC-γ1-mediated cellular proliferation independent its enzymatic activity. These results can provide novel insights to understand how PLC-γ1 is regulated and involved in cellular growth and proliferation.     

Protein kinase Calpha translocates to the perinuclear region to activate phospholipase D1

The inhibition of phorbol ester activation of phospholipase D1 (PLD1) by protein kinase C (PKC) inhibitors has been considered proof of phosphorylation-dependent activation of PLD1 by PKCalpha. We studied the effect of the PKC inhibitors Ro-31-8220 and bisindolylmaleimide I on PLD1 activation and found that they inhibited the activation by interfering with PKCalpha binding to PLD1. Further studies showed that only unphosphorylated PKCalpha could bind to and activate PLD1 and that both inhibitors induced phosphorylation of PKCalpha. The phosphorylation status of either PLD1 or PKCalpha per se did not affect PLD1 activation in vitro. Immunofluorescence studies showed that PLD1 remained in the perinuclear region after phorbol ester treatment, whereas PKCalpha translocated from cytosol to both plasma membrane and perinuclear regions. Both Ro-31-8220 and bisindolylmaleimide I blocked the translocation of PKCalpha to the perinuclear region but not to the plasma membrane. Studies with okadaic acid suggested that phosphorylation regulated the relocation of PKCalpha from the plasma membrane to the perinuclear region. It is proposed that localization and interaction of PKCalpha with PLD1 in the perinuclear region is required for PLD1 activation and that PKC inhibitors inhibit this through phosphorylation of PKCalpha, which blocks its translocation.     

The involvement of lipid activators of protein kinase C in the induction of ZIF268 in PC12 cells exposed to lead

Lead (Pb) induces the expression of immediate early genes (IEG) in PC12 cells by a mechanism that involves protein kinase C (PKC). To define the mechanisms, the involvement of two commonly observed lipid activators of PKC, diacylglycerols, and phosphatidylinositols, were examined. A dose-dependent increase in the expression of the IEG zif268 was observed in PC12 cells exposed to Pb. The PKC inhibitor Ro-31-8220 blocked the induction. An increase in levels of diacylglycerols was observed in PC12 cells exposed to Pb, but the increase was inhibited by Ro-31-8220. The phosphatidylinositol 3-kinase inhibitor Wortmannin, but not the inhibitor LY 294002, blocked the induction zif268 in Pb-exposed cells. Small increases in phosphatidylinositol 3-kinase activity were observed after exposure to Pb. In summary, diacylglycerols are elevated in PC12 cells exposed to Pb by a mechanism that requires PKC. It is possible that diacylglycerols contribute to the induction of zif268 by Pb by sustaining PKC activation.     

Comparison of PDGF-AA- and PDGF-BB-Induced Phosphoinositide Formation in Human and Mouse Fibroblasts

In certain cells, such as human fibroblasts (AG 1523), there is a clear difference in the cell motility response induced by the different isoforms of platelet-derived growth factor (PDGF). PDGF-BB induces extensive actin reorganization and is a potent chemotactic agent, whereas PDGF-AA has a limited effect on actin reorganization and is not chemotactic. In the present study, we wanted to compare these effects on cell motility with the effects of the PDGF isoforms on phosphoinositide (PtdIns) turnover. We find that stimulation of serum-starved AG 1523 cells with PDGF-AA or PDGF-BB caused an initial increase of the phosphatidylinositol phosphate and bisphosphate (PtdInsP and PtdInsP₂) pools, suggesting that activation of the phosphoinositide kinases is an initial response to PDGF stimulation. Despite a lower number of PDGF α-receptors than β-receptors on these cells, the initial formation of PtdInsP and PtdInsP₂ appears to be stimulated to a similar degree by the two PDGF isoforms. In contrast, PtdInsP₂ hydrolysis, indirectly measured as formation of phosphatidic acid, was correlated to the number of receptors. During prolonged exposure to PDGF-BB the stimulated PtdIns turnover remained at a high level, whereas the effect of PDGF-AA appeared more transient. A marked increase in the synthesis of a component migrating as phosphatidylinositol trisphosphate (PtdInsP_a) was also detected after stimulation with PDGF-BB for 5 min. With PDGF-AA minor amounts were found, indicating that activation of the PtdIns 3′-kinase occurs also via the PDGF α-receptor. Stimulation with PDGF-BB, but not -AA, also induced a 50% decrease in lyso-PtdIns. In murine fibroblasts (Swiss 3T3), where the two PDGF isoforms have a similar effect on cell motility, the two PDGF isoforms also similarly induced PtdIns turnover, PtdInsP₃ formation, and a decrease in lyso-PtdIns. Thus, there seems to be a correlation between PDGF-induced PtdIns turnover and PDGF-induced actin reorganization. This is compatible with previous evidence suggesting the microfilament formation is directly linked to an increased turnover of polyphosphoinositides in stimulated cells.     

ET-1 stimulates ERK signaling pathway through sequential activation of PKC and Src in rat myometrial cells

In this study, we analyzed in ratmyometrial cells the signaling pathways involved in the endothelin(ET)-1-induced extracellular signal-regulated kinase (ERK) activationrequired for the induction of DNA synthesis. We found that inhibitionof protein kinase C (PKC) by Ro-31-8220 abolished ERK activation.Inhibition of phospholipase C (PLC) by U-73122 or of phosphoinositide(PI) 3-kinase by wortmannin partially reduced ERK activation. A similarpartial inhibition was observed after treatment with pertussis toxin orPKC downregulation by phorbol ester treatment. The effect of wortmanninwas additive with that produced by PKC downregulation but not with thatdue to pertussis toxin. These results suggest that bothdiacylglycerol-sensitive PKC, activated by PLC products, anddiacylglycerol-insensitive PKC, possibly activated by aG_i-PI 3-kinase-dependent process, are involved inET-1-induced ERK activation. These two pathways were found to beactivated mainly through the ET_A receptor subtype. ET-1 andphorbol ester stimulated Src activity in a PKC-dependent manner, bothresponses being abolished in the presence of Ro-31-8220. Inhibition of Src kinases by PP1 abrogated phorbol ester- and ET-1-induced ERK activation. Finally, ET-1 activated Ras in a PP1-and Ro-31-8220-sensitive manner. Altogether, our results indicatethat ET-1 induces ERK activation in rat myometrial cells through thesequential stimulation of PKC, Src, and Ras.

     

Regulation of T cell receptor- and CD28-induced tyrosine phosphorylation of the focal adhesion tyrosine kinases Pyk2 and Fak by protein kinase C. A role for protein tyrosine phosphatases

The T cell receptor (TCR)-CD3 complex and the costimulatory molecule CD28 are critical for T cell function. Both receptors utilize protein tyrosine kinases (PTKs) for the phosphorylation of various signaling molecules, a process that is critical for the function of both receptors. The PTKs of the focal adhesion family, Pyk2 and Fak, have been implicated in the signaling of TCR and CD28. We show here evidence for the regulation of TCR- and CD28-induced tyrosine phosphorylation of the focal adhesion PTKs by protein kinase C (PKC). Thus, treating Jurkat T cells with the PKC activator phorbol 12-myristate 13-acetate (PMA) rapidly and strongly reversed receptor-induced tyrosine phosphorylation of the focal adhesion PTKs. In contrast, PMA did not affect TCR-induced tyrosine phosphorylation of CD3zeta or the PTKs Fyn and Zap-70. However, PMA induced a strong and rapid dephosphorylation of the linker molecule for activation of T cells. PMA failed to induce the dephosphorylation of proteins in PKC-depleted cells or in cells pretreated with the PKC inhibitor Ro-31-8220, confirming the role of PKC in mediating the PMA effect on receptor-induced protein tyrosine phosphorylation. The involvement of protein tyrosine phosphatases (PTPases) in mediating the dephosphorylation of the focal adhesion PTKs was confirmed by the failure of PMA to dephosphorylate Pyk2 in cells pretreated with the PTPase inhibitor orthovanadate. These results implicate PKC in the regulation of receptor-induced tyrosine phosphorylation of the focal adhesion PTKs in T cells. The data also suggest a role for PTPases in the PKC action.     

Angiotensin II-induced delayed stimulation of phospholipase C gamma1 requires activation of both phosphatidylinositol 3-kinase gamma and tyrosine kinase in vascular myocytes

In vascular smooth muscles, angiotensin II (AII) has been reported to activate phospholipase C (PLC) and phosphatidylinositol 3-kinase (PI3K). We investigated the time-dependent effects of AII on both phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) and inositol phosphates (InsPs) accumulation in permeabilized microsomes from rat portal vein smooth muscle in comparison with those of noradrenaline (NA). AII stimulated an early production of PtdInsP3 (within 30 s) followed by a delayed production of InsPs (within 3-5 min), in contrast to NA which activated only a fast production of InsPs. The use of pharmacological inhibitors and antibodies raised against the PI3K and PLC isoforms expressed in portal vein smooth muscle showed that AII specifically activated PI3Kgamma and that this isoform was involved in the AII-induced stimulation of InsPs accumulation. NA-induced InsPs accumulation depended on PLCbeta1 activation whereas AII-induced InsPs accumulation depended on PLCgamma1 activation. AII-induced PLCgamma1 activation required both tyrosine kinase and PI3Kgamma since genistein and tyrphostin B48 (inhibitors of tyrosine kinase), LY294002 and wortmannin (inhibitors of PI3K) and anti-PI3Kgamma antibody abolished AII-induced stimulation of InsPs accumulation. Increased tyrosine phosphorylation of PLCgamma1 was only detected for long-lasting applications of AII and was suppressed by genistein. These data indicate that activation of both PI3Kgamma and tyrosine kinase is a prerequisite for AII-induced stimulation of PLCgamma1 in vascular smooth muscle and suggest that the sequential activation of the three enzymes may be responsible for the slow and long-lasting contraction induced by AII.     

Protein kinase C-alpha and protein kinase C-epsilon are required for Grb2-associated binder-1 tyrosine phosphorylation in response to platelet-derived growth factor

Grb2-associated binder-1 (Gab1) is an adapter protein related to the insulin receptor substrate family. It is a substrate for the insulin receptor as well as the epidermal growth factor (EGF) receptor and other receptor-tyrosine kinases. To investigate the role of Gab1 in signaling pathways downstream of growth factor receptors, we stimulated rat aortic vascular smooth muscle cells (VSMC) with EGF and platelet-derived growth factor (PDGF). Gab1 was tyrosine-phosphorylated by EGF and PDGF within 1 min. AG1478 (an EGF receptor kinase-specific inhibitor) failed to block PDGF-induced Gab1 tyrosine phosphorylation, suggesting that transactivated EGF receptor is not responsible for this signaling event. Because Gab1 associates with phospholipase Cgamma (PLCgamma), we studied the role of the PLCgamma pathway in Gab1 tyrosine phosphorylation. Gab1 tyrosine phosphorylation by PDGF was impaired in Chinese hamster ovary cells expressing mutant PDGFbeta receptor (Y977F/Y989F: lacking the binding site for PLCgamma). Pretreatment of VSMC with (a specific PLCgamma inhibitor) inhibited Gab1 tyrosine phosphorylation as well, indicating the importance of the PLCgamma pathway. Gab1 was tyrosine-phosphorylated by phorbol ester to the same extent as PDGF stimulation. Studies using antisense protein kinase C (PKC) oligonucleotides and specific inhibitors showed that PKCalpha and PKCepsilon are required for Gab1 tyrosine phosphorylation. Binding of Gab1 to the protein-tyrosine phosphatase SHP2 and phosphatidylinositol 3-kinase was significantly decreased by PLCgamma and/or PKC inhibition, suggesting the importance of the PLCgamma/PKC-dependent Gab1 tyrosine phosphorylation for the interaction with other signaling molecules. Because PDGF-mediated ERK activation is enhanced in Chinese hamster ovary cells that overexpress Gab1, Gab1 serves as an important link between PKC and ERK activation by PDGFbeta receptors in VSMC.     

Regulation of PDGF-stimulated SHIP2 tyrosine phosphorylation and association with Shc in 3T3-L1 preadipocytes

In 3T3-L1 and human preadipocytes, insulin results in the isolated rise in phosphatidylinositol (PI)-3,4,5-P3, whereas PDGF produces PI(3,4)P2 in addition to PI(3,4,5)P3. SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) converts PI(3,4,5)P3 into PI(3,4)P2. PDGF, but not insulin, stimulates SHIP2 tyrosine phosphorylation and its association with Shc in human and 3T3-L1 preadipocytes. We now demonstrate that SHIP2 tyrosine phosphorylation and association with Shc in PDGF-treated 3T3-L1 preadipocytes was reduced by bisindolylmaleimide I (BisI), an inhibitor of conventional/novel protein kinase C (PKC). However, the production of PI(3,4)P2 and PI(3,4,5)P3 by PDGF was unaffected by BisI. Activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) was not sufficient to induce SHIP2 tyrosine phosphorylation. Furthermore, we identified threonine 958 (T958) as a novel PDGF-responsive SHIP2 phosphorylation site. Mutation of T958 to alanine reduced PDGF-stimulated SHIP2 tyrosine phosphorylation and association with Shc, but did not alter its anti-proliferative effect on preadipocytes. This study demonstrates that SHIP2 tyrosine phosphorylation and Shc association can be regulated by serine/threonine signaling pathways, either indirectly (via PKC), or directly (via T958). Interestingly, the anti-proliferative effect of SHIP2 T958A, as well as another SHIP2 mutant (Y986F, Y987F) that also displays defective tyrosine phosphorylation and Shc association, does not depend on these molecular events.     

Receptor tyrosine kinases regulate α1D-adrenoceptor signaling properties: Phosphorylation and desensitization

Human α_1D-adrenoceptors (truncated at the amino terminus (Δ1–79) to increase their membrane expression) were stably expressed in Rat-1 fibroblasts (1–1.5 pmol/mg protein). The receptors were functional as evidenced by a robust increase in intracellular calcium in response to noradrenaline. Using this cell line, the possibility that activation of receptor tyrosine kinases could modulate this adrenoceptor subtype was studied. It was observed that cell preincubation with insulin, IGF-I, EGF or PDGF markedly reduced the intracellular calcium increase observed in response to noradrenaline. Inhibitors of PI3K and PKC essentially blocked insulin-, IGF-I- and EGF-induced desensitizations. Interestingly, PDGF-induced α_1D-adrenergic desensitization was only partially ameliorated by PI3K inhibitors and was not affected by those of PKC. Insulin, IGF-I, EGF and PDGF induced concentration-dependent increases in the phosphorylation state of α_1D-adrenoceptors; phosphorylation took place on serine residues. Inhibitors of PI3K and PKC markedly reduced the effects of insulin, IGF-I and EGF on this parameter. These inhibitors only marginally reduced PDGF-induced α_1D-adrenoceptors phosphorylation. The ability of IGF-I to induce α_1D-adrenergic desensitization and phosphorylation was confirmed in cells expressing non-truncated rat α_1D-adrenenoceptors. Our data indicate that the function and phosphorylation state of α_1D-adrenoceptors is modulated by activation of receptor tyrosine kinases. Insulin, IGF-I and EGF actions take place through the action of PI3K and PKC; additional pathway(s) seem to participate in PDGF-induced α_1D-adrenoceptor desensitization and phosphorylation.     

Protein kinase Calpha mediates feedback inhibition of EGF receptor transactivation induced by Gq-coupled receptor agonists

While a great deal of attention has been focused on G-protein-coupled receptor (GPCR)-induced epidermal growth factor receptor (EGFR) transactivation, it has been known for many years that the tyrosine kinase activity of the EGFR is inhibited in cells treated with tumor-promoting phorbol esters, a process termed EGFR transmodulation. Because many GPCR agonists that elicit EGFR transactivation also stimulate the G_q/phospholipase C (PLC)/protein kinase C (PKC) pathway, we hypothesized that PKC-mediated inhibition of EGFR transactivation operates physiologically as a feedback loop that regulates the intensity and/or duration of GPCR-elicited EGFR transactivation. In support of this hypothesis, we found that treatment of intestinal epithelial IEC-18 cells with the PKC inhibitors GF 109203X or Ro 31-8220 or chronic exposure of these cells to phorbol-12,13-dibutyrate (PDB) to downregulate PKCs, markedly enhanced the increase in EGFR tyrosine phosphorylation induced by angiotensin II or vasopressin in these cells. Similarly, PKC inhibition enhanced EGFR transactivation in human colonic epithelial T84 cells stimulated with carbachol, as well as in bombesin-stimulated Rat-1 fibroblasts stably transfected with the bombesin receptor. Furthermore, cell treatment with inhibitors with greater specificity towards PKCα,  including Gö6976, Ro 31-7549 or Ro 32-0432, also increased GPCR-induced EGFR transactivation in IEC-18, T84 and Rat-1 cells. Transfection of siRNAs targeting PKCα  also enhanced bombesin-induced EGFR tyrosine phosphorylation in Rat-1 cells. Thus, multiple lines of evidence support the hypothesis that conventional PKC isoforms, especially PKCα, mediate feedback inhibition of GPCR-induced EGFR transactivation.     

Inhibition of platelet-derived growth factor receptor tyrosine kinase and downstream signaling pathways by Compound C

AMP-activated protein kinase (AMPK) has been implicated in anti-proliferative actions in a range of cell systems. Recently, it was observed that Compound C, an inhibitor of AMPK, also reduced the cell viability in human diploid fibroblasts (HDFs). Compound C-induced growth arrest was associated with a decrease in the cell cycle regulatory proteins, such as proliferating cell nuclear antigen, phosphorylated pRB, cyclin-dependent protein kinases (Cdk 2 and 4), cyclins (D and E), and the Cdk inhibitors (p21, p16, and p27). Therefore, the present study examined the molecular mechanism of the antiproliferative effects of Compound C. Although Compound C inhibited serum-induced phosphorylation of Akt and its substrate, glycogen synthase kinase-3β, it did not affect the Akt activity in vitro. Compound C significantly inhibited the receptor tyrosine phosphorylation and the activity of downstream signaling molecules, such as p85 phosphoinositide 3-kinase, phospholipase C-γ1, and extracellular signal-regulated kinase 1/2, induced by platelet-derived growth factor (PDGF) but not by epidermal growth factor- and insulin-like growth factor. In vitro growth factor receptor tyrosine kinase activity profiling revealed the IC₅₀ for PDGF receptor-β (PDGFRβ) to be 5.07 μM, whereas the IC₅₀ for the epidermal growth factor receptor and insulin-like growth factor receptor was ≥ 100 μM. The inhibitory effect of Compound C on PDGFRβ and Akt was also observed in AMPKα₁/α₂-knockout mouse embryonic fibroblasts, indicating that its inhibitory effect is independent of the AMPK activity. The inhibitory effect of Compound C on cell proliferation and PDGFRβ tyrosine phosphorylation was also demonstrated in various PDGFR-expressing cells, including MRC-5, BEAS-2B, rat aortic vascular smooth muscle cells, and A172 glioblastoma cells. These results indicate that Compound C can be used as a potential antiproliferative agent for PDGF- or PDGFR-associated diseases, such as cancer, atherosclerosis, and fibrosis.