Selective inhibition of tyrosine protein kinase by a synthetic multisubstrate analog

Synthetic compounds were designed in an attempt to mimic the possible transition state of tyrosine protein kinases. One representative compound (RP 53801) inhibited the enzyme purified from RSV-transformed cells. A serine/threonine kinase (kinase C) was 45 fold less sensitive. The inhibition was competitive with respect to ATP and noncompetitive with respect to the phosphate acceptor poly glu4-tyr1. The degree of inhibition (IC50 = 22 microM) was however lower than that expected from a transition state analog. The compound was capable of reducing tyrosine protein kinase activity in intact cells with some selectivity at 100 microM.     

In situ regulation of cell-cell communication by the cAMP-dependent protein kinase and protein kinase C

The effects of cAMP-dependent protein kinase A and protein kinase C on cell-cell communication have been examined in primary ovarian granulosa cells microinjected with purified components of these two regulatory cascades. These cells possess connexin43 ( 1)-type gap junctions, and are well-coupled electrotonically and as judged by the cell-to-cell transfer of fluorescent dye. Within 2–3 min after injection of the protein kinase A inhibitor (PKI) communication was sharply reduced or ceased, but resumed in about 3 min with the injection of the protein kinase A catalytic subunit. A similar resumption also occurred in PKI-injected cells after exposure to follicle stimulating hormone. Microinjection of the protein kinase C inhibitor protein caused a transient cessation of communication that spontaneously returned within 15–20 min. Treatment of cells with activators of protein kinase C, TPA or OAG for 60 min caused a significant reduction in communication that could be restored within 2–5 min by the subsequent injection of either the protein kinase C inhibitor or the protein kinase A catalytic subunit. With a longer exposure to either protein kinase C activator communication could not be restored and this appeared to be related to the absence of aggregates of connexin43 in membrane as detected immunologically. In cells injected with alkaline phosphatase communication stopped but returned either spontaneously within 20 min or within 2–3 min of injecting the cell with either the protein kinase A catalytic subunit or with protein kinase C. When untreated cells were injected with protein kinase C communication diminished or ceased within 5 min. Collectively these results demonstrate that cell-cell communication is regulated by both protein kinase A and C, but in a complex interrelated manner, quite likely by multiple phosphorylation of proteins within or regulating connexin-43 containing gap junctions.Abbreviations C catalytic subunit of protein kinase A - CKI protein kinase C inhibitor protein - Cx connexin protein - dbcAMP N⁶,2-O-dibutyryladenosine 3:5-cyclic monophosphate - OAG 1-oleoyl-2-acetyl-sn-glycerol - protein kinase A cAMP-dependent protein kinase - protein kinase C Ca²⁺-sensitive phospholipid-dependent protein kinase - PKI protein kinase A inhibitor protein - R regulatory subunit of protein kinase A - TRA 12-O-tetradecanoylphorbol-13-acetate - 8Br-cAMP 8-bromoadenosine 3:5 cyclic monophosphate     

Peroxynitrite-induced tyrosine nitration and inhibition of protein kinase C

Protein kinase C (PKC) is an important intracellular signaling molecule whose activity is essential for a number of aspects of neuronal function including synaptic plasticity. We investigated the regulation of PKC activity by reactive nitrogen species in order to examine whether such species regulate PKC in neurons. Neither autonomous nor cofactor-dependent PKC activity was altered when either hippocampal homogenates or rat brain purified PKC were incubated briefly with three different nitric oxide donor compounds. However, brief incubation of either hippocampal homogenates or purified PKC with peroxynitrite (ONOO(-)) inhibited cofactor-dependent PKC activity in a manner that correlated with the nitration of tyrosine residues on PKC, suggesting that this modification was responsible for the inhibition of PKC. Consistent with this idea, reducing agents had no effect on the inhibition of PKC activity caused by ONOO(-). Because there are numerous PKC isoforms that differ in the composition of the regulatory domain, we studied the effect of ONOO(-) on various PKC isoforms. ONOO(-) inhibited the cofactor-dependent activity of the alpha, betaII, epsilon, and zeta isoforms, indicating that inhibition of enzymatic activity by ONOO(-) was not PKC isoform-specific. We also were able to isolate nitrated PKCalpha and PKCbetaII from ONOO(-)-treated hippocampal homogenates via immunoprecipitation. Collectively, our findings support the hypothesis that ONOO(-) inhibits PKC activity via tyrosine nitration in neurons.     

Inhibition of EGF-induced phospholipase C activation in A431 cells by erbstatin, a tyrosine kinase inhibitor

Erbstatin, a tyrosine kinase inhibitor, inhibited epidermal growth factor (EGF)-induced inositol phosphate production in cultured A431 cells. However, it did not inhibit ATP-induced inositol phosphate production. Cytosolic but not membrane-associated phospholipase C was activated by EGF, and erbstatin inhibited enhancement of the phospholipase C activity in EGF-treated cells. Thus, tyrosine kinase of A431 cells is suggested to be functionally involved in phospholipase C activation.     

Mechanism of inhibition of cyclic nucleotide-gated channel by protein tyrosine kinase probed with genistein

Rod cyclic nucleotide-gated (CNG) channels are modulated by changes in tyrosine phosphorylation catalyzed by protein tyrosine kinases (PTKs) and phosphatases (PTPs). We used genistein, a PTK inhibitor, to probe the interaction between the channel and PTKs. Previously, we found that in addition to inhibiting tyrosine phosphorylation of the rod CNG channel alpha-subunit (RETalpha), genistein triggers a noncatalytic inhibitory interaction between the PTK and the channel. These studies suggest that PTKs affects RETalpha channels in two ways: (1) by catalyzing phosphorylation of the channel protein, and (2) by allosterically regulating channel activation. Here, we study the mechanism of noncatalytic inhibition. We find that noncatalytic inhibition follows the same activity dependence pattern as catalytic modulation (phosphorylation): the efficacy and apparent affinity of genistein inhibition are much higher for closed than for fully activated channels. Association rates with the genistein-PTK complex were similar for closed and fully activated channels and independent of genistein concentration. Dissociation rates were 100 times slower for closed channels, which is consistent with a much higher affinity for genistein-PTK. Genistein-PTK affects channel gating, but not single channel conductance or the number of active channels. By analyzing single channel gating during genistein-PTK dissociation, we determined the maximal open probability for normal and genistein-PTK-bound channels. genistein-PTK decreases open probability by increasing the free energy required for opening, making opening dramatically less favorable. Ni(2+), which potentiates RETalpha channel gating, partially relieves genistein inhibition, possibly by disrupting the association between the genistein-PTK and the channel. Studies on chimeric channels containing portions of RETalpha, which exhibits genistein inhibition, and the rat olfactory CNG channel alpha-subunit, which does not, reveals that a domain containing S6 and flanking regions is the crucial for genistein inhibition and may constitute the genistein-PTK binding site. Thus, genistein-PTK stabilizes the closed state of the channel by interacting with portions of the channel that participate in gating.     

Regulation of receptor tyrosine kinase signaling by protein tyrosine phosphatases

Signaling through receptor tyrosine kinases (RTKs) is a major mechanism for intercellular communication during development and in the adult organism, as well as in disease-associated processes. The phosphorylation status and signaling activity of RTKs is determined not only by the kinase activity of the RTK but also by the activities of protein tyrosine phosphatases (PTPs). This review discusses recently identified PTPs that negatively regulate various RTKs and the role of PTP inhibition in ligand-induced RTK activation. The contributions of PTPs to ligand-independent RTK activation and to RTK inactivation by other classes of receptors are also surveyed. Continued investigation into the involvement of PTPs in RTK regulation is likely to unravel previously unrecognized layers of RTK control and to suggest novel strategies for interference with disease-associated RTK signaling.     

Label-free electrochemical measurement of protein tyrosine kinase activity and inhibition based on electro-catalyzed tyrosine signaling

A novel label-free electrochemical method for measuring the activity of protein tyrosine kinases (PTK) has been developed. Epidermal growth factor receptor (EGFR), a typical PTK associated with a large percentage of all solid tumors, was used as the model kinase. Poly(glu, tyr) (4:1) peptide, as a substrate of EGFR, was covalently immobilized on the surface of indium tin oxide (ITO) electrode by silane chemistry. The tyrosine (Tyr) residue in the polypeptide served as an electrochemical signal reporter. Its voltammetric current was catalyzed by a dissolved electron mediator Os(bpy)(3)(2+) (bpy=2,2'-bipyridine) for increased sensitivity. Phosphorylation of the Tyr led to a loss of its electrochemical current, thus providing a sensing mechanism for PTK activity. Experimental conditions for the silanization of ITO surface and immobilization of polypeptide were investigated in details to facilitate the generation of Tyr electrochemical signal. The proposed biosensor exhibited high sensitivity and excellent stability. The limit of detection for EGFR was 1 UmL(-1). Furthermore, this biosensor can also be used for quantitative analysis of kinase inhibition. On the basis of the inhibitor concentration dependent electrochemical signal, the half-maximal inhibition value IC(50) of three EGFR inhibitors, PD-153035, OSI-774 and ZD-1839, and their corresponding inhibition constants K(i) were estimated, which were in agreement with those obtained from the conventional kinase assay. This electrochemical biosensor can be implemented in an array format for the high throughput assay of in vitro PTK activity and PTK inhibitors screening for practical diagnostic application and drug discovery.     

In situ phosphorylation of platelet actin-binding protein by cAMP-dependent protein kinase stabilizes it against proteolysis by calpain

To identify the protein kinase that is responsible for catalyzing phosphorylation of actin-binding protein (ABP) in platelets, we have examined the effects of protein kinase C and cAMP-dependent protein kinase on this process. We found that purified platelet protein kinase C from platelets was unable to phosphorylate ABP in vitro. However, a crude platelet kinase preparation phosphorylated ABP in the presence of cAMP, but not in the presence of Ca2+/phosphatidylserine. Fresh platelet plasma membranes incubated with [gamma-32P]ATP phosphorylated ABP in the presence of cAMP and the process was blocked by a cAMP-dependent protein kinase inhibitor; ABP phosphorylation induced by prostaglandin E1 (PGE1) appeared to be reduced by the subsequent addition of thrombin. These results strongly suggest that in situ ABP is phosphorylated by activated cAMP-dependent protein kinase when platelet function is inhibited by PGE1. Furthermore, in the PGE1-treated platelets, ABP was proteolyzed at a slower rate than in control platelets when they were lysed with Triton in the absence of EGTA. Partially purified ABP was proteolyzed by calpain in vitro at a slower rate as well. It was demonstrated that ABP from PGE1-treated platelets recovered its sensitivity to calpain after ABP was incubated with a protein phosphatase that had been purified from platelets. We postulate that ABP is stabilized against proteolysis in response to cAMP-elevating agents and that this blocks cytoskeleton reorganization.     

Nuclear localization of Lyn tyrosine kinase mediated by inhibition of its kinase activity

Src-family kinases, cytoplasmic enzymes that participate in various signaling events, are found at not only the plasma membrane but also subcellular compartments, such as the nucleus, the Golgi apparatus and late endosomes/lysosomes. Lyn, a member of the Src-family kinases, is known to play a role in DNA damage response and cell cycle control in the nucleus. However, it is still unclear how the localization of Lyn to the nucleus is regulated. Here, we investigated the mechanism of the distribution of Lyn between the cytoplasm and the nucleus in epitheloid HeLa cells and hematopoietic THP-1 cells. Lyn was definitely detected in purified nuclei by immunofluorescence and immunoblotting analyses. Nuclear accumulation of Lyn was enhanced upon treatment of cells with leptomycin B (LMB), an inhibitor of Crm1-mediated nuclear export. Moreover, Lyn mutants lacking the sites for lipid modification were highly accumulated in the nucleus upon LMB treatment. Intriguingly, inhibition of the kinase activity of Lyn by SU6656, Csk overexpression, or point mutation in the ATP-binding site induced an increase in nuclear Lyn levels. These results suggest that Lyn being imported into and rapidly exported from the nucleus preferentially accumulates in the nucleus by inhibition of the kinase activity and lipid modification.     

Protein tyrosine kinase but not protein kinase C inhibition blocks receptor induced alveolar macrophage activation

The selective enzyme inhibitors genistein and Ro 31-8220 were used to assess the importance of protein tyrosine kinase (PTK) and protein kinase C (PKC), respectively, in N-formyl-methionyl-leucyl-phenylalanine (FMLP) induced generation of superoxide anion and thromboxane B(2) (TXB(2)) in guinea-pig alveolar macrophages (AM). Genistein (3-100 muM) dose dependently inhibited FMLP (3 nM) induced superoxide generation in non-primed AM and TXB(2) release in non-primed or in lipopolysaccharide (LPS) (10 ng/ml) primed AM to a level > 80% but had litle effect up to 100 muM on phorbol myristate acetate (PMA) (10 nM) induced superoxide release. Ro 31-8220 inhibited PMA induced superoxide generation (IC(50) 0.21 +/- 0.10 muM) but had no effect on or potentiated (at 3 and 10 muM) FMLP responses in non-primed AM. In contrast, when present during LPS priming as well as during FMLP challenge Ro 31-8220 (10 muM) inhibited primed TXB(2) release by > 80%. The results indicate that PTK activation is required for the generation of these inflammatory mediators by FMLP in AM. PKC activation appears to be required for LPS priming but not for transducing the FMLP signal; rather, PKC activation may modulate the signal by a negative feedback mechanism.     

Regulation of receptor tyrosine kinase signaling by protein tyrosine phosphatase-1B

Receptor tyrosine kinases (RTKs) are key regulators of cellular homeostasis. Based on in vitro and ex vivo studies, protein tyrosine phosphatase-1B (PTP1B) was implicated in the regulation of several RTKs, yet mice lacking PTP1B show defects mainly in insulin and leptin receptor signaling. To address this apparent paradox, we studied RTK signaling in primary and immortalized fibroblasts from PTP1B(-/-) mice. After growth factor treatment, cells lacking PTP1B exhibit increased and sustained phosphorylation of the epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor (PDGFR). However, Erk activation is enhanced only slightly, and there is no increase in Akt activation in PTP1B-deficient cells. Our results show that PTP1B does play a role in regulating EGFR and PDGFR phosphorylation but that other signaling mechanisms can largely compensate for PTP1B deficiency. In-gel phosphatase experiments suggest that other PTPs may help to regulate the EGFR and PDGFR in PTP1B(-/-) fibroblasts. This and other compensatory mechanisms prevent widespread, uncontrolled activation of RTKs in the absence of PTP1B and probably explain the relatively mild effects of PTP1B deletion in mice.     

Functional characterization of peanut serine/threonine/tyrosine protein kinase: molecular docking and inhibition kinetics with tyrosine kinase inhibitors

Serine/threonine/tyrosine (STY) protein kinase from peanut is developmentally regulated and is induced by abiotic stresses. In addition, STY protein kinase activity is regulated by tyrosine phosphorylation. Kinetic mechanism of plant dual specificity protein kinases is not studied so far. Recombinant STY protein kinase occurs as a monomer in solution as shown by gel filtration chromatography. The relative phosphorylation rate of kinase against increasing enzyme concentrations follows a first-order kinetics indicating an intramolecular phosphorylation mechanism. Moreover, the active recombinant STY protein kinase could not transphosphorylate a kinase-deficient mutant of STY protein kinase. Molecular docking studies revealed that the tyrosine kinase inhibitors bind the protein kinase at the same region as ATP. STY protein kinase activity was inhibited by the tyrosine kinase inhibitors, and the inhibitor potency series against the recombinant STY protein kinase was tyrphostin > genistein > staurosporine. The inhibition constant (K(i)), and the IC(50) value of STY protein kinase for tyrosine kinase inhibitors with ATP and histone are discussed. All the inhibitors competed with ATP. Genistein was an uncompetitive inhibitor with histone, whereas staurosporine and tyrphostin were linear mixed type noncompetitive inhibitors with histone. Molecular docking and kinetic analysis revealed that Y148F mutant of the "ATP-binding loop" and Y297F mutant of the "activation loop" showed a dramatic increase in K(i) values for genistein and tyrphostin with respect to wild-type STY protein kinase. Data presented here provide the direct evidence on the mechanism of inhibition of plant protein kinases by tyrosine kinase inhibitors. This study also suggests that tyrosine kinase inhibitors may be useful in unraveling the plant tyrosine phosphorylation signaling cascades.     

Stimulation of tyrosine protein kinase activity by dimethyl sulfoxide

Dimethyl sulfoxide (DMSO) stimulated the activity of a partially purified tyrosine protein kinase from rat lung. The stimulation was concentration dependent with a maximum stimulation (about 2 fold) observed at 10 per cent (V/V) DMSO. On the other hand, acetone (10 percent, V/V), did not exert any stimulatory effect on the enzyme activity. The stimulation was associated with a decrease in the Km for the substrate and an increase in the Vmax. In contrast, the Km for ATP was not affected by DMSO. Under identical assay conditions, DMSO did not significantly alter the activities of phosphorylase kinase, catalytic subunit of cAMP-dependent protein kinase and Ca2+-phospholipid-dependent protein kinase. It may be speculated that stimulation of tyrosine protein kinase may be one of the mechanisms by which DMSO exerts its biological effects.     

Characterization of fodrin phosphorylation by spleen protein tyrosine kinase

Fodrin, an actin and calmodulin binding and spectrin-like protein present in many nonerythrocyte tissues, could be phosphorylated up to more than 1.5 mol of phosphate/mol of protein by a highly purified non-receptor-associated protein tyrosine kinase from bovine spleen. The protein phosphorylation was not affected by Ca2+/calmodulin or by F-actin. Km and Vmax values of the reaction were 91 nM and 0.35 nmol of P2 min-1 (mg of kinase)-1, respectively. Both subunits A and B of fodrin were phosphorylated, with the rate of subunit A phosphorylation much greater than that of subunit B phosphorylation. Tryptic phosphopeptide mapping of the phosphorylated subunits suggested that there were three major phosphorylation sites in subunit A and one in subunit B. Phosphotyrosylfodrin could be dephosphorylated by the calmodulin-stimulated phosphatase (calcineurin) in the presence of activating metal ions; Ni2+ was a much more effective activator than Mn2+ for this reaction. Fodrin phosphorylation by the spleen protein tyrosine kinase did not appear to alter the actin and calmodulin binding properties of the protein. On the other hand, the calmodulin-dependent stimulation of smooth muscle actomyosin Mg2+-ATPase by fodrin was enhanced by 101% +/- 3% (n = 3) upon fodrin phosphorylation. Ni2+-calcineurin, which was shown to effectively dephosphorylate the phosphotyrosyl residues on fodrin, could reverse the phosphorylation-enhanced Mg2+-ATPase stimulatory activity of fodrin.     

Inhibition of phosphorylase kinase, and tyrosine protein kinase activities by quercetin

Quercetin, a naturally occurring bioflavonoid inhibited the activities of phosphorylase kinase and a partially purified tyrosine protein kinase from rat lung. The inhibition was rapid and concentration dependent. Quercetin at 100 microM inhibited the activities of phosphorylase kinase and tyrosine protein kinase by about 95 and 80-90 percent respectively. ATP reversed the quercetin mediated inhibition of tyrosine protein kinase but not of phosphorylase kinase. These data suggest that quercetin has differential effect on different protein kinase activities and it may be used as a tool to probe the role of various protein kinases in cell function.     

In situ phosphorylation of human platelet myosin heavy and light chains by protein kinase C

Treatment of human platelets with 162 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in phosphorylation of a number of peptides, including myosin heavy chain and the 20-kDa myosin light chain. The site phosphorylated on the myosin heavy chain was localized by two-dimensional peptide mapping to a serine residue(s) in a single major tryptic phosphopeptide. This phosphopeptide co-migrated with a tryptic peptide that was produced following in vitro phosphorylation of platelet myosin heavy chain using protein kinase C. The sites phosphorylated in the 20-kDa myosin light chain in intact cells were analyzed by two-dimensional mapping of tryptic peptides and found to correspond to Ser1 and Ser2 in the turkey gizzard myosin light chain. In vitro phosphorylation of purified human platelet myosin by protein kinase C showed that in addition to Ser1 and Ser2, a third site corresponding to Thr9 in turkey gizzard myosin light chain is also phosphorylated. The phosphorylatable myosin light chains from human platelets were found to consist of two major isoforms present in approximately equal amounts, but differing in their molecular weights and isoelectric points. A third, minor isoform was also visualized by two-dimensional gel electrophoresis. Following treatment with TPA, both the mono- and diphosphorylated forms of each isoform could be visualized, and the sites of phosphorylation were identified. The phosphate content rose from negligible amounts found prior to treatment with TPA to 1.2 mol of phosphate/mol of myosin light chain and 0.7 mol of phosphate/mol of myosin heavy chain following treatment. These results suggest that TPA mediates phosphorylation of both myosin light and heavy chains in intact platelets by activation of protein kinase C.     

The inhibition of EGF-dependent proliferation of keratinocytes by tyrphostin tyrosine kinase blockers

Protein tyrosine kinase blockers of the tyrphostin family inhibited the EGF-dependent proliferation of human and guinea pig keratinocytes grown in culture and induced their growth arrest. These blockers also significantly inhibited the growth of epidermal keratinocytes, but not of dermal cells, in whole skin organ culture from both guinea pig and human origin. The antiproliferative activity of these tyrphostins correlated quantitatively with their potency as inhibitors of EGF receptor autophosphorylation and the EGF-dependent protein phosphorylation of intracellular target proteins in the keratinocyte. Furthermore, no significant cell cytotoxicity or reduction in serine and threonine phosphorylation of many intracellular polypeptides were observed upon incubation of the cells with tyrphostins like AG213. The complete growth arrest induced by the tyrphostins is fully reversible and upon their removal the keratinocytes resumed their growth with the original growth rate. Because of the nontoxic nature of these compounds and their growth-arresting properties, we suggest their use as agents to treat hyperproliferative conditions of human skin.     

Copurification of tyrosine hydroxylase from rat pheochromocytoma by protein kinase

Rat pheochromocytoma contains a protein kinase activity which remains associated with tyrosine hydroxylase (TH) during its purification. The incorporation of phosphate in TH is observed after incubation of TH with labelled ATP and magnesium without the need for an exogenous protein kinase. This Ca2+ and cAMP-independent kinase activity is different from previously described TH phosphorylating kinases from rat pheochromocytoma and other tissues.     

Direct inhibition of the hexose transporter GLUT1 by tyrosine kinase inhibitors

The facilitative hexose transporter GLUT1 is a multifunctional protein that transports hexoses and dehydroascorbic acid, the oxidized form of vitamin C, and interacts with several molecules structurally unrelated to the transported substrates. Here we analyzed in detail the interaction of GLUT1 with a group of tyrosine kinase inhibitors that include natural products of the family of flavones and isoflavones and synthetic compounds such as the tyrphostins. These compounds inhibited, in a dose-dependent manner, the transport of hexoses and dehydroascorbic acid in human myeloid HL-60 cells, in transfected Chinese hamster ovary cells overexpressing GLUT1, and in normal human erythrocytes, and blocked the glucose-displaceable binding of cytochalasin B to GLUT1 in erythrocyte ghosts. Kinetic analysis of transport data indicated that only tyrosine kinase inhibitors with specificity for ATP binding sites inhibited the transport activity of GLUT1 in a competitive manner. In contrast, those inhibitors that are competitive with tyrosine but not with ATP failed to inhibit hexose uptake or did so in a noncompetitive manner. These results, together with recent evidence demonstrating that GLUT1 is a nucleotide binding protein, support the concept that the inhibitory effect on transport is related to the direct interaction of the inhibitors with GLUT1. We conclude that predicted nucleotide-binding motifs present in GLUT1 are important for the interaction of the tyrosine kinase inhibitors with the transporter and may participate directly in the binding transport of substrates by GLUT1.