Cell growth inhibition by a novel vitamin K is associated with induction of protein tyrosine phosphorylation

We have shown that a synthetic vitamin K analog, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone or compound 5 (Cpd 5), potently inhibits cell growth and suggested that the analog exerts its effects mainly via sulfhydryl arylation rather than redox cycling. Since protein-tyrosine phosphatases (PTPases), which have pivotal roles in many cellular functions, have a critical cysteine in their active site, we have proposed PTPases as likely targets for Cpd 5. To test this hypothesis, we examined the effects of Cpd 5 on protein tyrosine phosphorylation of cellular proteins and on the activity of PTPases. We found that Cpd 5 rapidly induced protein tyrosine phosphorylation in a human hepatocellular carcinoma cell line (Hep3B) at growth inhibitory doses, and the effect was blocked by thiols but not by non-thiol antioxidants or tyrosine kinase inhibitors. Cpd 5 inhibited PTPase activity, which was also significantly antagonized by reduced glutathione. Furthermore, the well studied PTPase inhibitor orthovanadate also induced protein tyrosine phosphorylation and growth inhibition in Hep3B cells. These results suggest that inhibition of cellular PTPases by sulfhydryl arylation and subsequent perturbation of protein tyrosine phosphorylation may be involved in the mechanisms of Cpd 5-induced cell growth inhibition.     

Cell to substratum adhesion is involved in v-Src-induced cellular protein tyrosine phosphorylation: implication for the adhesion-regulated protein tyrosine phosphatase activity

Protein tyrosine phosphorylation accompanies the integrin-mediated cell to substratum adhesion, and is essential for the progression of G1/S phase of the cell-cycle in normal fibroblasts. To examine how cellular protein tyrosine phosphatase (PTPase) activity is involved in regulating the adhesion-dependent protein tyrosine phosphorylation, we employed fibroblast cells bearing an active form of a protein tyrosine kinase (PTK), v-Src. We found that the v-Src induced tyrosine phosphorylation in certain proteins such as tensin, talin, p120, p80/85 (cortactin) and paxillin was greatly reduced when the cell to substratum adhesion was lost. Readhesion of the cells onto fibronectin restored these phosphorylation events, while this was inhibited by the addition of RGD peptide. The kinase activity of the v-Src was unchanged by the loss of cell to substratum adhesion. On the other hand, treatment with a protein tyrosine phosphatase inhibitor vanadate caused much the same increase in the v-Src-mediated cellular tyrosine phosphorylation between cells adhered to the culture environments and cells kept in suspension. These data suggest that PTPase(s) appears to be more critical than the v-Src PTK in determining the cell adhesion-dependent protein tyrosine phosphorylation. Moreover, most of the protein tyrosine phosphorylations that are mediated by the v-Src but still dependent on the cell adhesion were indeed greatly reduced during an anchorage-independent growth of v-Src cells. Thus our data collectively indicate that the v-Src induced high level of tyrosine phosphorylation in certain types of proteins are still under the control of the integrin(s) or the cell adhesion to culture substratum, and most of these adhesion-regulated high levels of tyrosine phosphorylations are not essential for the transformed phenotype.     

Purification, identification and subcellular distribution of three predominant protein-tyrosine phosphatase enzymes in skeletal muscle tissue

Protein-tyrosine phosphatases (PTPases) play a key role in the regulation of insulin action. In order to identify PTPases in skeletal muscle, the major site of insulin-mediated glucose disposal in vivo, we purified PTPases from rat muscle tissue fractions by a series of column chromatographic techniques. PTPase activities were assayed by measuring the dephosphorylation of a rat insulin receptor kinase domain, derivatized lysozyme and p-nitrophenylphosphate, and the enzymes were further characterized by immunoblotting. Of the total PTPase activity in muscle homogenates, 51-64% was localized to the solubilized particulate fraction, with the specific PTPase activity 3.3-fold and 5.6-fold higher in the particulate fraction towards RCM-lysozyme or the insulin receptor, respectively. The major peak (> 75%) of PTPase activity in the particulate fraction was purified further to 700-fold; 75% of this activity passed through a Blue-3GA column and revealed immunoreactivity for both LAR and SH-PTP2. PTPase activity retained on the Blue-3GA column contained PTPase1B. The major peak (> 70%) from muscle cytosol was further purified to 1500-fold. After the Blue-3GA step, immunoblotting revealed both SH-PTP2 and PTPase1B in the cytosol fraction, but LAR was absent from this fraction. LRP (RPTP-alpha) was not detected by blotting the PTPase activities from the purified particulate or cytosol fractions. Immunodepletion studies demonstrated that LAR, SH-PTP2 and PTPase1B were quantitatively major PTPase activities in the initial muscle homogenate, together accounting for over 70% of the total activity towards RCM-lysozyme. These studies provide insight into the relative abundance and subcellular distribution of specific PTPases in muscle tissue that are involved in the regulation of reversible tyrosine phosphorylation in this tissue.     

The transmembrane protein-tyrosine phosphatase CD45 is associated with decreased insulin receptor signaling

Overexpression of the transmembrane protein-tyrosine phosphatase (PTPase) CD45 in nonhematopoietic cells results in decreased signaling through growth factor receptor tyrosine kinases. Consistent with these data, insulin receptor signaling is increased when the CD45-related PTPase LAR is reduced by antisense suppression in a rat hepatoma cell line. To test whether the hematopoietic cell-specific PTPase CD45 functions in a manner similar to LAR by negatively modulating insulin receptor signaling in hematopoietic cells, the insulin-responsive human multiple myeloma cell line U266 was isolated into two subpopulations that differed in CD45 expression. In CD45 nonexpressing (CD45-) cells, insulin receptor autophosphorylation was increased by 3-fold after insulin treatment when compared to CD45 expressing (CD45+) cells. This increase in receptor autophosphorylation was associated with similar increases in insulin-dependent tyrosine kinase activation. These receptor level effects were paralleled by postreceptor responses. Insulin-dependent tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) and Shc was 3-fold greater in CD45- cells. In addition, insulin-dependent IRS-1/phosphatidylinositol 3-kinase association and MAP kinase activation in CD45- cells were also 3-fold larger. While expression of CD45 was associated with a decrease in the responsiveness of early insulin receptor signaling, interleukin 6-dependent activation of mitogen-activated protein kinase kinase and mitogen-activated protein kinase was equivalent between CD45- and CD45+ cells. These observations indicate that CD45 can function as a negative modulator of growth factor receptor tyrosine kinases in addition to its well-established role as an activator of src family tyrosine kinases.     

The second domain of the CD45 protein tyrosine phosphatase is critical for interleukin-2 secretion and substrate recruitment of TCR-zeta in vivo

The CD45 protein tyrosine phosphatase (PTPase) has been shown to regulate the activity of Lck and Fyn protein tyrosine kinases in T cells. However, it is not clear that these constitute the only CD45 substrates. Moreover, the manner by which PTPase activity and substrate recruitment are regulated, is poorly understood. Previous in vitro studies suggest that the first cytoplasmic PTPase domain (D1) of CD45 is the active PTPase, which may be regulated by an enzymatically inactive second PTPase domain (D2). However, the function of CD45 D2 in vivo is unknown. In this study, reconstitution of CD45(-) T cells with specific CD45 PTPase mutants allowed demonstration of a critical role for D2 in TCR-mediated interleukin (IL)-2 production. Specifically, replacement of CD45 D2 with that of the LAR PTPase to form a CD45/LAR:D2 chimera, abrogates CD45-dependent IL-2 production. This effect cannot be accounted for by loss of PTPase activity per se. The expression of D1 substrate-trapping mutants reveals an in vivo interaction between CD45 and TCR-zeta that is dependent on CD45 D2. Thus, cells expressing CD45 lacking D2 exhibit abnormal TCR-mediated signaling characterized by hyperphosphorylation of zeta and deficient ZAP-70 phosphorylation. These data suggest an essential role for CD45 D2 in TCR-regulated IL-2 production through substrate recruitment of the zeta chain.     

From Vanadis to Atropos: vanadium compounds as pharmacological tools in cell death signalling

Vanadium compounds exert a variety of biological responses, the most notable being their effects as insulin mimetics. More recently, they have been used as pharmacological tools to investigate signalling pathways. Some peroxovanadium compounds act as powerful protein tyrosine phosphatase inhibitors, modulating both the extent and duration of phosphotyrosine signals at the level of the transmembrane growth factor receptors and targets in the cytoplasm and nucleus. A brief history of vanadium compounds, selected chemical properties of vanadium compounds and the ability of peroxovanadium complexes to modulate the activities of protein tyrosine phosphatases and tyrosine kinases are presented in this review by Anne Morinville, Dusica Maysinger and Alan Shaver. From the range of biological activities of these compounds, this review focuses on cytotoxic effects and possible roles of mitogen-activated protein kinases in mediating the effects exerted by vanadium compounds.     

Activation in vitro of somatostatin receptor subtypes 2, 3, or 4 stimulates protein tyrosine phosphatase activity in membranes from transfected Ras-transformed NIH 3T3 cells: coexpression with catalytically inactive SHP-2 blocks responsiveness

Somatostatin receptors (sstr) subtypes 1-5 were transiently expressed in NIH 3T3 cells stably transformed with Ha-Ras(G12V) to assess the ability of each receptor to stimulate protein tyrosine phosphatase (PTPase) activity in vitro. Treatment of membranes from sstr2-, sstr3-, or sstr4-expressing cells with somatostatin-14 plus guanyl-5'-yl imidodiphosphate (GMPPNP) increased PTPase activity, and this stimulation was pertussis toxin-sensitive. Somatostatin alone, GMPPNP alone, or somatostatin plus GDP were ineffective under these conditions. sstr1 and sstr5 failed to increase PTPase activity although both receptors were expressed, as assessed by appearance of high-affinity binding sites for [125I-Tyr11]somatostatin-14. Somatostatin plus GMPPNP stimulated PTPase activity in vitro when sstr2 was coexpressed with wild type PTP1B or a Cys to Ser (C/S), catalytically inactive PTP1B or with wild type SH2-domain containing PTPase SHP-2. However, coexpression with catalytically inactive C/S SHP-2 abrogated this response. Thus, three of the five cloned sstr's can couple to activate PTPase in this cellular background. Abrogation of the response by C/S SHP-2 strongly suggests, but does not prove, a role for SHP-2 in the mechanism.     

Protein tyrosine phosphatase inhibitors in Fc gamma RI-induced myeloid oxidant signaling

Fc-receptor stimulation in myeloid cells results in increased oxygen consumption, termed the respiratory burst, which is coupled to a rapid and transient increase in tyrosine phosphorylation of cellular proteins. In a previous paper in this journal we showed that the protein tyrosine phosphatase (PTPase) inhibitors sodium orthovanadate and phenylarsine oxide (PAO) block the Fc gamma RI-induced respiratory burst in interferon-gamma-differentiated U937 cells (U937IF) while augmenting the Fc gamma RI-induced tyrosine phosphorylation of cellular proteins. Herein we examine the effects of PTPase inhibitors on specific molecules involved in Fc gamma RI signaling. We show that orthovanadate and PAO augmented the Fc gamma RI-induced tyrosine phosphorylation of the adaptor protein CBL. CBL interactions with other phosphoproteins, among them SHC and CRKL, were also augmented in response to pretreatment with the PTPase inhibitors. SHC was tyrosine phosphorylated in response to Fc gamma RI stimulation of U937IF cells and bound to the SH2 domain of GRB2 in a stimulation-dependent manner. In fusion protein pull-down experiments the interaction of SHC with the SH2 domain of GRB2 was increased in PTPase inhibitor pretreated U937IF cells in response to Fc gamma RI stimulation. Our data support the hypothesis that a tyrosine dephosphorylation event is required for effective transmission of the Fc gamma RI signal to result in activation of the myeloid respiratory burst response.     

Regulation of adenylyl cyclase activity in human peripheral blood mononuclear cells: effects of protein kinase inhibitors and of a calcium ionophore

In a previous paper we presented evidence for a negative regulation of adenylyl cyclase activity by tyrosine protein kinase(s) in the human leukemic T cell line Jurkat. In order to examine this point in non malignant cells, we conducted the present study in human peripheral blood mononuclear cells (PBMC). In these cells, staurosporine, a broad spectrum protein kinase inhibitor, enhanced not only the receptor-mediated, induced by prostaglandin E2 (PGE2), but also the direct (forskolin-induced) stimulation of adenylyl cyclase activity. Herbimycin A, a specific protein tyrosine kinase inhibitor, reproduced only in part the effect of staurosporine, whereas bisindolylmaleimide, the most specific protein kinase C (PKC) inhibitor known at present time, was ineffective. All these observations were made both in the absence and presence of isobutylmethylxanthine, a phosphodiesterase inhibitor, indicating that the effects of staurosporine and herbimycin A on cAMP accumulation were not due to phosphodiesterase inhibition. The calcium ionophore A 23187 also enhanced the PGE2-induced cAMP accumulation, and this effect was not additive to that of staurosporine, but additive to that of herbimycin A. These results confirm and extend those obtained in Jurkat cells. Taken together, they indicate that in human PBMC the adenylyl cyclase activity is negatively regulated by tyrosine kinase(s) and not by PKC, and positively regulated by Ca2+. They also suggest that the major enhancement by staurosporine of the PGE2-induced cAMP accumulation, although chiefly mediated by protein tyrosine kinase inhibition, also depends on another, presently undetermined, effect of the drug simulating that of Ca2+.     

Decrease in protein tyrosine phosphatase activities in vanadate-treated obese Zucker (fa/fa) rat liver

The inhibitory action of vanadate towards protein tyrosine phosphatase (PTPase) has been considered as a probable mechanism by which it exerts insulin-like effects. In this study, we have examined the in vivo effects of vanadate on PTPases in the liver of obese Zucker rats, a genetic animal model for obesity and type II diabetes. These animals were characterized by hyperinsulinemia and mild hyperglycemia. The number of insulin receptors were significantly (p < 0.01) decreased in liver. After chronic administration of vanadate in obese rats, 80% decrease in the plasma levels of insulin was observed. The insulin receptor numbers were significantly (p < 0.01) higher in vanadate-treated obese rats as compared to the untreated ones. The hepatic PTPase activities in cytosolic and particulate fractions, with phosphorylated poly glu:tyr (4:1) and the insulin receptor peptide (residues 1142-1153) as substrates, increased in obese rats. In vanadate-treated obese rat livers, the PTPase activities in both subcellular fractions with these substrates decreased significantly (p < 0.001). The decreases in PTPase activities from these groups of rats were further supported by chromatography on a Mono Q column. These data support the view that inhibition of PTPases plays a role in the insulin-mimetic action of vanadate.     

Inhibitory and stimulatory effects of somatostatin on two human pancreatic cancer cell lines: a primary role for tyrosine phosphatase SHP-1

Somatostatin (SS-14) and its structural analogue SMS 201-995 (SMS) are recognized as physiological inhibitors of multiple organs and tissue functions through specific membrane receptors (sst1-sst5). The effects of SS-14 and SMS in the growth control of the pancreatic cancer cell lines MIA PaCa-2 and PANC-1 were investigated to identify and clarify the intracellular events involved. In PANC-1 cells, SS-14 and SMS caused inhibition of their basal growth, and that stimulated by epidermal growth factor, with a maximal effect at 0.1-1 microM. To understand the inhibitory mechanisms, we investigated the effects of SS-14 and SMS on phosphotyrosine phosphatase (PTPase) activity and, more specifically, that of tyrosine phosphatase SHP-1 (PTP1C). SS-14 and SMS caused significant increases in total cellular PTPase activity, and particularly SHP-1, with maximal activation within 1 min. Inhibition of membrane tyrosine kinase and p42 MAP kinase activities was also observed, in response to SS-14 and SMS. In MIA PaCa-2 cells, SS-14 and SMS were associated with a positive growth response at 1-10 nM, after 4 days of culture in serum-free medium. Total cellular PTPase activity was slightly increased, but SHP-1 activity could not be detected; its absence in this cell line was confirmed by Western blot. Membrane tyrosine kinase activities were significantly increased by SS-14 and SMS at concentrations needed for maximal growth. p44/p42, which are constitutively active in this cell line, and p38 activities were not affected by somatostatin. In conclusion, somatostatin can exert different effects on human pancreatic cancer cell growth, depending upon the presence or absence of SHP-1. This enzyme can play a key role in the control of cell proliferation, and its cellular presence may determine the therapeutic potential of somatostatin in the control of cancer cell growth.     

Tyrosine kinase inhibitors suppress prostaglandin F2alpha-induced phosphoinositide hydrolysis, Ca2+ elevation and contraction in iris sphincter smooth muscle

We investigated the effects of the protein tyrosine kinase inhibitors, genistein, tyrphostin 47, and herbimycin on prostaglandin F2alpha- and carbachol-induced inositol-1,4,5-trisphosphate (IP3) production, [Ca2+]i mobilization and contraction in cat iris sphincter smooth muscle. Prostaglandin F2alpha and carbachol induced contraction in a concentration-dependent manner with EC50 values of 0.92 x 10(-9) and 1.75 x 10(-8) M, respectively. The protein tyrosine kinase inhibitors blocked the stimulatory effects of prostaglandin F2alpha, but not those evoked by carbachol, on IP3 accumulation, [Ca2+]i mobilization and contraction, suggesting involvement of protein tyrosine kinase activity in the physiological actions of the prostaglandin. Daidzein and tyrphostin A, inactive negative control compounds for genistein and tyrphostin 47, respectively, were without effect. Latanoprost, a prostaglandin F2alpha analog used as an antiglaucoma drug, induced contraction and this effect was blocked by genistein. Genistein (10 microM) markedly reduced (by 67%) prostaglandin F2alpha-stimulated increase in [Ca2+]i but had little effect on that of carbachol in cat iris sphincter smooth muscle cells. Vanadate, a potent inhibitor of protein tyrosine phosphatase, induced a slow gradual muscle contraction in a concentration-dependent manner with an EC50 of 82 microM and increased IP3 generation in a concentration-dependent manner with an EC50 of 90 microM. The effects of vanadate were abolished by genistein (10 microM). Wortmannin, a myosin light chain kinase inhibitor, reduced prostaglandin F2alpha- and carbachol-induced contraction, suggesting that the involvement of protein tyrosine kinase activity may lie upstream of the increases in [Ca2+]i evoked by prostaglandin F2alpha. Further studies aimed at elucidating the role of protein tyrosine kinase activity in the coupling mechanism between prostaglandin F2alpha receptor activation and increases in intracellular Ca2+ mobilization and identifying the tyrosine-phosphorylated substrates will provide important information about the role of protein tyrosine kinase in the mechanism of smooth muscle contraction, as well as about the mechanism of the intraocular pressure lowering effect of the prostaglandin in glaucoma patients.     

The tumor-selective somatostatin analog, TT2-32 induces a biphasic activation of phosphotyrosine phosphatase activity in human colon tumor cell line, SW620

Somatostatin has been demonstrated to activate phosphotyrosine phosphatases (PTPases) in pancreatic cells. In this work we studied the effect of a tumor-selective somatostatin structural derivative, TT2-32, on the PTPase activity in the SW620 human colon tumor cell line. TT2-32 caused a strong inhibition of cell proliferation. In response to TT2-32 we found a rapid and sustained increase (5-30 min) in PTPase activity showing two maxima at 0.1 and 30 microM concentrations, respectively. During short-term incubation tyrosine kinase activity was much less affected by TT2-32. TT2-32-induced activation of PTPases may be an important early step in the signaling cascade in the inhibition of cell proliferation in colon carcinomas.     

Crystal structure of an angiogenesis inhibitor bound to the FGF receptor tyrosine kinase domain

Angiogenesis, the sprouting of new blood vessels from pre-existing ones, is an essential physiological process in development, yet also plays a major role in the progression of human diseases such as diabetic retinopathy, atherosclerosis and cancer. The effects of the most potent angiogenic factors, vascular endothelial growth factor (VEGF), angiopoietin and fibroblast growth factor (FGF) are mediated through cell surface receptors that possess intrinsic protein tyrosine kinase activity. In this report, we describe a synthetic compound of the pyrido[2,3-d]pyrimidine class, designated PD 173074, that selectively inhibits the tyrosine kinase activities of the FGF and VEGF receptors. We show that systemic administration of PD 173074 in mice can effectively block angiogenesis induced by either FGF or VEGF with no apparent toxicity. To elucidate the determinants of selectivity, we have determined the crystal structure of PD 173074 in complex with the tyrosine kinase domain of FGF receptor 1 at 2.5 A resolution. A high degree of surface complementarity between PD 173074 and the hydrophobic, ATP-binding pocket of FGF receptor 1 underlies the potency and selectivity of this inhibitor. PD 173074 is thus a promising candidate for a therapeutic angiogenesis inhibitor to be used in the treatment of cancer and other diseases whose progression is dependent upon new blood vessel formation.     

Prolactin-mediated inhibition of 20alpha-hydroxysteroid dehydrogenase gene expression and the tyrosine kinase system

The rat luteal 20alpha-hydroxysteroid dehydrogenase plays a key role at catabolizing progesterone and at decreasing the level of this steroid secreted by the ovaries. Throughout pregnancy and before parturition neither the mRNA nor the protein for this enzyme could be detected. In this investigation we set to examine whether PRL and PRL-like hormone from placental origin silence the expression of this gene and whether PRL action involves tyrosine kinase activity and/or de novo protein synthesis. The results revealed that PRL and PRL-like hormone from rat placental origin (rPL-1 and rPL-2), but not rat growth hormone, caused a rapid and profound inhibition of 20alpha-HSD mRNA expression in highly luteinized granulosa cells. Immunoprecipition and western blot analysis indicate that PRL-R associates with JAK2 and Stat5, and this association is increased within 30 seconds with PRL treatment. Although both JAK2 and Stat5 were phosphorylated on tyrosine upon PRL treatment, the PRL mediated inhibition of 20alpha-HSD was not reversed by either tyrosine kinase inhibitors, AG18 and genistein, but was largely reversed by the protein synthesis inhibitor cycloheximide. In summary, results of this investigation indicate that although PRL can activate the JAK2/Stat5 system in the corpus luteum, the down regulation of 20alpha-HSD mRNA by PRL does not appear to involve tyrosine kinase activity but depends on de novo synthesis of protein(s).     

GLEPP1, a renal glomerular epithelial cell (podocyte) membrane protein-tyrosine phosphatase. Identification, molecular cloning, and characterization in rabbit

Podocytes are specialized epithelial cells with delicate interdigitating foot processes which cover the exterior basement membrane surface of the glomerular capillary. They are in part responsible for the extraordinary charge and size filtration characteristics of the glomerulus. To better understand disease processes affecting the glomerular filter, we searched for proteins with relative specificity to the podocyte using a monoclonal antibody strategy. The first such protein characterized (designated glomerular epithelial protein 1 (GLEPP1)) is a membrane protein-tyrosine phosphatase (PTPase) with a large extracellular domain containing eight fibronectin type III-like repeats, a hydrophobic transmembrane segment, and a single PTPase domain. The GLEPP1 PTPase domain shows homology with two other single domain transmembrane PTPases (PTP beta and Drosophila central nervous system PTP10D). This homology includes 2 cysteines in the PTPase domain not present in intracellular or tandem domain membrane PTPases. GLEPP1 PTPase protein is distributed to the podocyte foot processes themselves. RNase protection assay shows that GLEPP1 mRNA is also present in brain. By analogy with the CD45 PTPase of T cells, we expect that this receptor might play a role in maintaining foot process structure and/or function by regulating tyrosine phosphorylation of podocyte proteins.     

Inhibition of nicotinic responses of bovine adrenal chromaffin cells by the protein kinase C inhibitor, Ro 31-8220

1. The effects of the protein kinase C inhibitor, Ro 31-8220, on the responses of cultured bovine adrenal chromaffin cells to nicotine, phorbol 12, 13-dibutyrate (PDBu) and K+ have been investigated. 2. Tyrosine hydroxylase activity was measured in situ in intact cells by measuring 14CO2 evolved following the hydroxylation and rapid decarboxylation of [14C]-tyrosine offered to the cells. Secretion of endogenous adrenaline and noradrenaline was measured by use of h.p.l.c. with electrochemical detection. Cyclic AMP levels were measured in cell extracts by RIA. 3. Ro 31-8220 produced a concentration-dependent inhibition of 300 nM PDBu-stimulated tyrosine hydroxylase activity with an IC50 of < 2 microM and complete inhibition at 10 microM. It had no effect on the responses to forskolin. 4. Ro 31-8220 produced a concentration-dependent inhibition of 5 microM nicotine-stimulated tyrosine hydroxylase activity, adrenaline and noradrenaline secretion and cellular cyclic AMP levels, with an IC50 of about 3 microM and complete inhibition by 10 microM. At concentrations up to 10 microM, Ro 31-8220 had little or no effect on the corresponding responses to 50 mm K+. 5. A structural analogue of Ro 31-8220, bisindolylmaleimide V, that lacks activity as a protein kinase C inhibitor, had no effect up to 10 microM on PDBu-stimulated tyrosine hydroxylase activity or on nicotine-stimulated cyclic AMP levels or noradrenaline secretion and only marginal inhibitory effects on nicotine-stimulated tyrosine hydroxylase activity and adrenaline secretion. 6. A structurally related protein kinase C inhibitor, bisindolylmaleimide I, inhibited PDBu-stimulated tyrosine hydroxylase activity with an IC50 of < 1 microM and complete inhibition by 3 microM, but had essentially no effect on nicotine stimulated tyrosine hydroxylase activity or catecholamine secretion. 7. The results suggest that Ro 31-8220 is not only a protein kinase C inhibitor but is also a potent inhibitor of nicotinic receptor responses in adrenal chromaffin cells by a mechanism unrelated to protein kinase C inhibition. The results are consistent with Ro 31-8220 being a nicotinic receptor antagonist.     

Platelet-derived growth factor-stimulated secretion of basement membrane proteins by skeletal muscle occurs by tyrosine kinase-dependent and -independent pathways

The basement membrane of skeletal muscle is produced by the muscle cells it ensheathes and by nonmuscle cells located in the surrounding extracellular matrix. In this study, we have shown that platelet-derived growth factor (PDGF) stimulates secretion of three basement membrane components of skeletal muscle: laminin (70% increase), fibronectin (30%), and type IV collagen (70%). Furthermore, we have found using the signal transduction inhibitors, genistein (tyrosine kinase inhibitor), phorbol 12-myristate 13-acetate (protein kinase C (PKC) inhibitor), thapsigargin (depletes intracellular Ca2+ stores), and H89 (protein kinase A inhibitor), that PDGF-stimulated secretion of these proteins occurs through distinct signaling pathways. Densitometry of Western blots of L6 myoblast supernatant indicates that the PDGF-induced increase in secretion of laminin and type IV collagen is tyrosine kinase-dependent. The increase in type IV collagen secretion also shows dependence on PKC, as well as the release of intracellular Ca2+. Inhibition of either of these pathways reduces the increase in type IV collagen secretion to 20%. In contrast, the PDGF-induced increase in laminin secretion is unaffected by inhibition of either PKC or intracellular Ca2+ release. The increase in fibronectin secretion by PDGF uses yet a third set of signals. PDGF-induced fibronectin secretion is not dependent on tyrosine kinase activity but is dependent on protein kinase A as well as the release of intracellular Ca2+. These divergent signaling pathways provide for independent regulation of basement membrane protein secretion, allowing a muscle cell to modify both the quantity and composition of its basement membrane in response to its environment.     

Phosphorylation of insulin receptor substrate 1 by glycogen synthase kinase 3 impairs insulin action

The phosphorylation of insulin receptor substrate 1 (IRS-1) on tyrosine residues by the insulin receptor (IR) tyrosine kinase is involved in most of the biological responses of insulin. IRS-1 mediates insulin signaling by recruiting SH2 proteins through its multiple tyrosine phosphorylation sites. The phosphorylation of IRS-1 on serine/threonine residues also occurs in cells; however, the particular protein kinase(s) promoting this type of phosphorylation are unknown. Here we report that glycogen synthase kinase 3 (GSK-3) is capable of phosphorylating IRS-1 and that this modification converts IRS-1 into an inhibitor of IR tyrosine kinase activity in vitro. Expression of wild-type GSK-3 or an "unregulated" mutant of the kinase (S9A) in CHO cells overexpressing IRS-1 and IR, resulted in increased serine phosphorylation levels of IRS-1, suggesting that IRS-1 is a cellular target of GSK-3. Furthermore, insulin-induced tyrosine phosphorylation of IRS-1 and IR was markedly suppressed in cells expressing wild-type or the S9A mutant, indicating that expression of GSK-3 impairs IR tyrosine kinase activity. Taken together, our studies suggest a new role for GSK-3 in attenuating insulin signaling via its phosphorylation of IRS-1 and may provide new insight into mechanisms important in insulin resistance.