Fibronectin fragment activation of proline-rich tyrosine kinase PYK2 mediates integrin signals regulating collagenase-3 expression by human chondrocytes through a protein kinase C-dependent pathway

Fibronectin fragments (FN-f), including the 110-kDa fragment that binds the alpha5beta1 integrin, stimulate collagenase-3 (MMP-13) production and cartilage destruction. In the present study, treatment of chondrocytes with the 110-kDa FN-f or an activating antibody to the alpha5beta1 integrin was found to increase tyrosine autophosphorylation (Tyr-402) of the proline-rich tyrosine kinase-2 (PYK2) without significant change in autophosphorylation (Tyr-397) of focal adhesion kinase (FAK). The tyrosine kinase inhibitor tyrphostin A9, shown previously to block a PYK2-dependent pathway, blocked the FN-f-stimulated increase in MMP-13, whereas tyrphostin A25 did not. FN-f-stimulated PYK2 phosphorylation and MMP-13 production was also blocked by reducing intracellular calcium levels. Adenovirally mediated overexpression of wild type but not mutant PYK2 resulted in increased MMP-13 production. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate stimulated PYK2 phosphorylation and MMP-13 production. MMP-13 expression stimulated by either phorbol 12-myristate 13-acetate or FN-f was blocked by PKC inhibitors including the PKCdelta inhibitor rottlerin. Furthermore, PKCdelta translocation from cytosol to membrane was noted within 5 min of stimulation with FN-f. Immortalized human chondrocytes, transiently transfected with MMP-13 promoter-luciferase reporter constructs, showed increased promoter activity after FN-f treatment that was inhibited by co-transfection with either of two dominant negative mutants of PYK2 (Y402F and K457A). No inhibition was seen after cotransfection with wild type PYK2, a dominant negative of FAK (FRNK) or empty vector plasmid. FN-f-stimulated MMP-13 promoter activity was also inhibited by chemical inhibitors of ERK, JNK, and p38 mitogen-activated protein (MAP) kinases or by co-transfection of dominant negative MAP kinase mutant constructs. These studies have identified a novel pathway for the MAP kinase regulation of MMP-13 production which involves FN-f stimulation of the alpha5beta1 integrin and activation of the nonreceptor tyrosine kinase PYK2 by PKC, most likely PKCdelta     

Rottlerin is a mitochondrial uncoupler that decreases cellular ATP levels and indirectly blocks protein kinase Cdelta tyrosine phosphorylation

Protein kinase Cdelta (PKCdelta) is activated by stimuli that increase its tyrosine phosphorylation, including neurotransmitters that initiate fluid secretion in salivary gland (parotid) epithelial cells. Rottlerin, a compound reported to be a PKCdelta-selective inhibitor, rapidly increased the rate of oxygen consumption (QO2) of parotid acinar cells and PC12 cells. In parotid cells, this was distinct from the effects of the muscarinic receptor ligand carbachol, which promoted a sodium pump-dependent increase in respiration. Rottlerin increased the QO2 of isolated rat liver mitochondria to a level similar to that produced when oxidative phosphorylation was initiated by ADP or when mitochondria were uncoupled by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The effects of rottlerin on mitochondrial QO2 were neither mimicked nor blocked by the PKC inhibitor GF109203X. Rottlerin was not effective in blocking PKCdelta activity in vitro. Exposure of freshly isolated parotid acinar cells to rottlerin and FCCP reduced cellular ATP levels and reduced stimuli-dependent increases in tyrosine phosphorylation of PKCdelta. Neither rottlerin nor FCCP reduced stimuli-dependent PKCdelta tyrosine phosphorylation in RPG1 cells (a salivary ductal line) or PC12 cells, consistent with their dependence on glycolysis rather than oxidative phosphorylation for energy-dependent processes. These results demonstrate that rottlerin directly uncouples mitochondrial respiration from oxidative phosphorylation. Previous studies using rottlerin should be evaluated cautiously.     

Modulation of PKCdelta tyrosine phosphorylation and activity in salivary and PC-12 cells by Src kinases

Protein kinase C (PKC) delta becomes tyrosine phosphorylated in rat parotid acinar cells exposed to muscarinic and substance P receptor agonists, which initiate fluid secretion in this salivary cell. Here we examine the signaling components of PKCdelta tyrosine phosphorylation and effects of phosphorylation on PKCdelta activity. Carbachol- and substance P-promoted increases in PKCdelta tyrosine phosphorylation were blocked by inhibiting phospholipase C (PLC) but not by blocking intracellular Ca2+ concentration elevation, suggesting that diacylglycerol, rather than D-myo-inositol 1,4,5-trisphosphate production, positively modulated this phosphorylation. Stimuli-dependent increases in PKCdelta activity in parotid and PC-12 cells were blocked in vivo by inhibitors of Src tyrosine kinases. Dephosphorylation of tyrosine residues by PTP1B, a protein tyrosine phosphatase, reduced the enhanced PKCdelta activity. Lipid cofactors modified the tyrosine phosphorylation-dependent PKCdelta activation. Two PKCdelta regulatory sites (Thr-505 and Ser-662) were constitutively phosphorylated in unstimulated parotid cells, and these phosphorylations were not altered by stimuli that increased PKCdelta tyrosine phosphorylation. These results demonstrate that PKCdelta activity is positively modulated by tyrosine phosphorylation in parotid and PC-12 cells and suggest that PLC-dependent effects of secretagogues on salivary cells involve Src-related kinases.     

Targeting of PYK2 to focal adhesions as a cellular mechanism for convergence between integrins and G protein-coupled receptor signaling cascades

The non-receptor tyrosine kinase PYK2 appears to function at a point of convergence of integrins and certain G protein-coupled receptor (GPCR) signaling cascades. In this study, we provide evidence that translocation of PYK2 to focal adhesions is triggered both by cell adhesion to extracellular matrix proteins and by activation of the histamine GPCR. By using different mutants of PYK2 as green fluorescent fusion proteins, we show that the translocation of PYK2 to focal adhesions is not dependent on its catalytic activity but rather is mediated by its carboxyl-terminal domain. Translocation of PYK2 to focal adhesions was attributed to enhanced tyrosine phosphorylation of PYK2 and its association with the focal adhesion proteins paxillin and p130(Cas). Translocation of PYK2 to focal adhesions, as well as its tyrosine phosphorylation in response to histamine treatment, was abolished in the presence of protein kinase C inhibitors or cytochalasin D treatment, whereas activation of protein kinase C by phorbol ester resulted in focal adhesion targeting of PYK2 and its tyrosine phosphorylation in an integrin-clustering dependent manner. Overexpression of a wild-type PYK2 enhanced ERK activation in response to histamine, whereas a kinase-deficient mutant substantially inhibited this response. Furthermore, inhibition of PYK2 translocation to focal adhesions abolished ERK activation in response to histamine treatment. These results suggest that PYK2 apparently links between GPCRs and focal adhesion-dependent ERK activation and can provide the molecular basis underlying PYK2 function at a point of convergence between signaling pathways triggered by extracellular matrix proteins and certain GPCR agonists.     

CD45 negatively regulates monocytic cell differentiation by inhibiting phorbol 12-myristate 13-acetate-dependent activation and tyrosine phosphorylation of protein kinase Cdelta

The protein-tyrosine phosphatase CD45 is expressed on all monocytic cells, but its function in these cells is not well defined. Here we report that CD45 negatively regulates monocyte differentiation by inhibiting phorbol 12-myristate 13-acetate (PMA)-dependent activation of protein kinase C (PKC) delta. We found that antisense reduction of CD45 in U937 monocytic cells (CD45as cells) increased by 100% the ability of PMA to enlarge cell size, increase cell cytoplasmic process width and length, and induce surface expression of CD11b. In addition, reduction in CD45 expression caused the duration of peak PMA-induced MEK and extracellular signal-regulated kinase (ERK) 1/2 activity to increase from 5 min to 30 min while leading to a 4-fold increase in PMA-dependent PKCdelta activation. Importantly, PMA-dependent tyrosine phosphorylation of PKCdelta was also increased 4-fold in CD45as cells. Finally, inhibitors of MEK (PD98059) and PKCdelta (rottlerin) completely blocked PMA-induced monocytic cell differentiation. Taken together, these data indicate that CD45 inhibits PMA-dependent PKCdelta activation by impeding PMA-dependent PKCdelta tyrosine phosphorylation. Furthermore, this blunting of PKCdelta activation leads to an inhibition of PKCdelta-dependent activation of ERK1/2 and ERK1/2-dependent monocyte differentiation. These findings suggest that CD45 is a critical regulator of monocytic cell development.     

Phosphorylation of protein kinase Cdelta on distinct tyrosine residues regulates specific cellular functions

Protein kinase Cdelta (PKCdelta) inhibits proliferation and decreases expression of the differentiation marker glutamine synthetase (GS) in C6 glioma cells. Here, we report that distinct, specific tyrosine residues on PKCdelta are involved in these two responses. Transfection of cells with PKCdelta mutated at tyrosine 155 to phenylalanine caused enhanced proliferation in response to 12-phorbol 12-myristate 13-acetate, whereas GS expression resembled that for the PKCdelta wild-type transfectant. Conversely, transfection with PKCdelta mutated at tyrosine 187 to phenylalanine resulted in increased expression of GS, whereas the rate of proliferation resembled that of the PKCdelta wild-type transfectant. The tyrosine phosphorylation of PKCdelta and the decrease in GS expression induced by platelet-derived growth factor (PDGF) were abolished by the Src kinase inhibitors PP1 and PP2. In response to PDGF, Fyn associated with PKCdelta via tyrosine 187. Finally, overexpression of dominant negative Fyn abrogated the decrease in GS expression and reduced the tyrosine phosphorylation of PKCdelta induced by PDGF. We conclude that the tyrosine phosphorylation of PKCdelta and its association with tyrosine kinases may be an important point of divergence in PKC signaling.     

Infection of glioma cells with Sindbis virus induces selective activation and tyrosine phosphorylation of protein kinase C delta. Implications for Sindbis virus-induced apoptosis

Sindbis virus (SV) is an alpha virus used as a model for studying the role of apoptosis in virus infection. In this study, we examined the role of protein kinase C (PKC) in the apoptosis induced by SVNI, a virulent strain of SV. Infection of C6 cells with SVNI induced a selective translocation of PKCdelta to the endoplasmic reticulum and its tyrosine phosphorylation. The specific PKCdelta inhibitor rottlerin and a PKCdelta kinase-dead mutant increased the apoptosis induced by SVNI. To examine the role of the tyrosine phosphorylation of PKCdelta in the apoptosis induced by SVNI we used a PKCdelta mutant in which five tyrosine residues were mutated to phenylalanine (PKCdelta5). PKCdelta5-overexpressing cells exhibited increased apoptosis in response to SVNI as compared with control cells and to cells overexpressing PKCdelta. SVNI also increased the cleavage of caspase 3 in cells overexpressing PKCdelta5 but did not induce cleavage of PKCdelta or PKCdelta5. Using single tyrosine mutants, we identified tyrosines 52, 64, and 155 as the phosphorylation sites associated with the apoptosis induced by SVNI. We conclude that PKCdelta exerts an inhibitory effect on the apoptosis induced by SV and that phosphorylation of PKCdelta on specific tyrosines is required for this function.     

Roles of tyrosine phosphorylation and cleavage of protein kinase Cdelta in its protective effect against tumor necrosis factor-related apoptosis inducing ligand-induced apoptosis

Protein kinase Cdelta (PKCdelta) regulates cell apoptosis in a cell- and stimulus-specific manner. Here, we studied the role of PKCdelta in the apoptotic effect of TRAIL in glioma cells. We found that transfection of the cells with a PKCdelta kinase-dead mutant (K376R) or with a small interfering RNA targeting the PKCdelta mRNA increased the apoptotic effect of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), whereas overexpression of PKCdelta decreased it. PKCdelta acted downstream of caspase 8 and upstream of cytochrome c release from the mitochondria. TRAIL induced cleavage of PKCdelta within 2-3 h of treatment, which was abolished by caspase 3, 8, and 9 inhibitors. The cleavage of PKCdelta was essential for its protective effect because overexpression of a caspase-resistant mutant (PKCdeltaD327A) did not protect glioma cells from TRAIL-induced apoptosis but rather increased it. TRAIL induced translocation of PKCdelta to the perinuclear region and the endoplasmic reticulum and phosphorylation of PKCdelta on tyrosine 155. Using a PKCdeltaY155F mutant, we found that the phosphorylation of PKCdelta on tyrosine 155 was essential for the cleavage of PKCdelta in response to TRAIL and for its translocation to the endoplasmic reticulum. In addition, phosphorylation of PKCdelta on tyrosine 155 was necessary for the activation of AKT in response to TRAIL. Our results indicate that PKCdelta protects glioma cells from the apoptosis induced by TRAIL and implicate the phosphorylation of PKCdelta on tyrosine 155 and its cleavage as essential factors in the anti-apoptotic effect of PKCdelta.     

Inhibition of amylase secretion from differentiated AR4-2J pancreatic acinar cells by an actin cytoskeleton controlled protein tyrosine phosphatase activity.

Disruption of the actin cytoskeleton in AR4-2J pancreatic acinar cells led to an increase in cytosolic protein tyrosine phosphatase activity, abolished bombesin-induced tyrosine phosphorylation and reduced bombesin-induced amylase secretion by about 45%. Furthermore, both tyrosine phosphorylation and amylase secretion induced by phorbol ester-induced activation of protein kinase C were abolished. An increase in the cytosolic free Ca2+ concentration by the Ca2+ ionophore A23187 had no effect on tyrosine phosphorylation but induced amylase release. Only when added together with phorbol ester, the same level of amylase secretion as with bombesin was reached. This amylase secretion was inhibited by about 40%, by actin cytoskeleton disruption similar to that induced by bombesin. We conclude that actin cytoskeleton-controlled protein tyrosine phosphatase activity downstream of protein kinase C activity regulates tyrosine phosphorylation which in part is involved in bombesin-stimulated amylase secretion.     

Depolarization activates ERK and proline-rich tyrosine kinase 2 (PYK2) independently in different cellular compartments in hippocampal slices

In the hippocampus, extracellular signal-regulated kinase (ERK) and the non-receptor protein proline-rich tyrosine kinase 2 (PYK2) are activated by depolarization and involved in synaptic plasticity. Both are also activated under pathological conditions following ischemia, convulsions, or electroconvulsive shock. Although in non-neuronal cells PYK2 activates ERK through the recruitment of Src-family kinases (SFKs), the link between these pathways in the hippocampus is not known. We addressed this question using K(+)-depolarized rat hippocampal slices. Depolarization increased the phosphorylation of PYK2, SFKs, and ERK. These effects resulted from Ca(2+) influx through voltage-gated Ca(2+) channels and were diminished by GF109203X, a protein kinase C inhibitor. Inhibition of SFKs with PP2 decreased PYK2 tyrosine phosphorylation dramatically, but not its autophosphorylation on Tyr-402. Moreover, PYK2 autophosphorylation and total tyrosine phosphorylation were profoundly altered in fyn-/- mice, revealing an important functional relationship between Fyn and PYK2 in the hippocampus. In contrast, ERK activation was unaltered by PP2, Fyn knock-out, or LY294002, a phosphatidyl-inositol-3-kinase inhibitor. ERK activation was prevented by MEK inhibitors that had no effect on PYK2. Immunofluorescence of hippocampal slices showed that PYK2 and ERK were activated in distinct cellular compartments in somatodendritic regions and nerve terminals, respectively, with virtually no overlap. Activation of ERK was critical for the rephosphorylation of a synaptic vesicle protein, synapsin I, following depolarization, underlining its functional importance in nerve terminals. Thus, in hippocampal slices, in contrast to cell lines, depolarization-induced activation of non-receptor tyrosine kinases and ERK occurs independently in distinct cellular compartments in which they appear to have different functional roles.     

Phorbol 12-myristate 13-acetate-dependent protein kinase C delta-Tyr311 phosphorylation in cardiomyocyte caveolae

Protein kinase Cdelta (PKCdelta) activation is generally attributed to lipid cofactor-dependent allosteric activation mechanisms at membranes. However, recent studies indicate that PKCdelta also is dynamically regulated through tyrosine phosphorylation in H(2)O(2)- and phorbol 12-myristate 13-acetate (PMA)-treated cardiomyocytes. H(2)O(2) activates Src and related Src-family kinases (SFKs), which function as dual PKCdelta-Tyr(311) and -Tyr(332) kinases in vitro and contribute to H(2)O(2)-dependent PKCdelta-Tyr(311)/Tyr(332) phosphorylation in cardiomyocytes and in mouse embryo fibroblasts. H(2)O(2)-dependent PKCdelta-Tyr(311)/Tyr(332) phosphorylation is defective in SYF cells (deficient in SFKs) and restored by Src re-expression. PMA also promotes PKCdelta-Tyr(311) phosphorylation, but this is not associated with SFK activation or PKCdelta-Tyr(332) phosphorylation. Rather, PMA increases PKCdelta-Tyr(311) phosphorylation by delivering PKCdelta to SFK-enriched caveolae. Cyclodextrin treatment disrupts caveolae and blocks PMA-dependent PKCdelta-Tyr(311) phosphorylation, without blocking H(2)O(2)-dependent PKCdelta-Tyr(311) phosphorylation. The enzyme that acts as a PKCdelta-Tyr(311) kinase without increasing PKCdelta phosphorylation at Tyr(332) in PMA-treated cardiomyocytes is uncertain. Although in vitro kinase assays implicate c-Abl as a selective PKCdelta-Tyr(311) kinase, PMA-dependent PKCdelta-Tyr(311) phosphorylation persists in cardiomyocytes treated with the c-Abl inhibitor ST1571 and c-Abl is not detected in caveolae; these results effectively exclude a c-Abl-dependent process. Finally, we show that 1,2-dioleoyl-sn-glycerol mimics the effect of PMA to drive PKCdelta to caveolae and increase PKCdelta-Tyr(311) phosphorylation, whereas G protein-coupled receptor agonists such as norepinephrine and endothelin-1 do not. These results suggest that norepinephrine and endothelin-1 increase 1,2-dioleoyl-sn-glycerol accumulation and activate PKCdelta exclusively in non-caveolae membranes. Collectively, these results identify stimulus-specific PKCdelta localization and tyrosine phosphorylation mechanisms that could be targeted for therapeutic advantage.     

Signal transduction of physiological concentrations of vasopressin in A7r5 vascular smooth muscle cells. A role for PYK2 and tyrosine phosphorylation of K+ channels in the stimulation of Ca2+ spiking.

The signal transduction pathway linking physiological concentrations of [Arg(8)]vasopressin (AVP) to an increase in frequency of Ca(2+) spiking was examined in confluent cultures of A7r5 vascular smooth muscle cells. Immunoprecipitation/Western blot studies revealed a robust increase in tyrosine phosphorylation of the non-receptor tyrosine kinase, PYK2, in A7r5 cells treated with 4beta-phorbol 12-myristate 13-acetate or ionomycin. 100 pm AVP also induced PYK2 tyrosine phosphorylation, and this effect was inhibited by protein kinase C inhibitors Ro-31-8220 (1-10 microm) or chelerythrine chloride (1-20 microm). In fura-2-loaded A7r5 cells, the stimulation of Ca(2+) spiking by 100 pm AVP or 1 nm 4beta-phorbol 12-myristate 13-acetate was completely blocked by PP2 (10 microm, a Src family kinase inhibitor). Salicylate (20 mm, recently identified as a PYK2 inhibitor) and the tyrosine kinase inhibitor, tyrphostin A47 (50 microm), but not its inactive analog, tyrphostin A63, also blocked AVP-stimulated Ca(2+) spiking. PYK2 phosphorylation was inhibited by both PP2 and salicylate, whereas tyrphostin A47 failed to inhibit PYK2 tyrosine phosphorylation. ERK1/2 kinases did not appear to be involved because 1) 100 pm AVP did not appreciably increase ERK1/2 phosphorylation and U-0126 (2.5 microm) did not inhibit AVP-stimulated Ca(2+) spiking; and 2) epidermal growth factor (10 nm) robustly stimulated ERK1/2 phosphorylation but did not induce Ca(2+) spiking. Delayed rectifier K(+) channels may mediate the PYK2 activity because Kv1.2 channel protein co-immunoprecipitated with PYK2 and tyrosine phosphorylation of Kv1.2 was stimulated by AVP and inhibited by Ro-31-8220, PP2, and salicylate but not tyrphostin A47. Our findings are consistent with a role for PYK2 and phosphorylation of K(+) channels in the stimulation of Ca(2+) spiking by physiological concentrations of AVP.     

Src tyrosine kinase regulates insulin-induced activation of protein kinase C (PKC) delta in skeletal muscle

Insulin stimulation of skeletal muscle results in rapid activation of protein kinase Cdelta (PKCdelta), which is associated with its tyrosine phosphorylation and physical association with insulin receptor (IR). The mechanisms underlying tyrosine phosphorylation of PKCdelta have not been determined. In this study, we investigated the possibility that the Src family of nonreceptor tyrosine kinases may be involved upstream insulin signaling. Studies were done on differentiated rat skeletal myotubes in primary culture. Insulin caused an immediate stimulation of Src and induced its physical association with both IR and PKCdelta. Inhibition of Src by treatment with the Src family inhibitor PP2 reduced insulin-stimulated Src-PKCdelta association, PKCdelta tyrosine phosphorylation and PKCdelta activation. PP2 inhibition of Src also decreased insulin-induced IR tyrosine phosphorylation, IR-PKCdelta association and association of Src with both PKCdelta and IR. Finally, inhibition of Src decreased insulin-induced glucose uptake. We conclude that insulin activates Src tyrosine kinase, which regulates PKCdelta activity. Thus, Src tyrosine kinase may play an important role in insulin-induced tyrosine phosphorylation of both IR and PKCdelta. Moreover, both Src and PKCdelta appear to be involved in IR activation and subsequent downstream signaling.     

Unique regulation of c-Jun N-terminal kinase by PYK2/CAK-beta in angiotensin II-stimulated vascular smooth muscle cells

Activation of tyrosine kinases is believed to play a central role in angiotensin II (AngII) signaling. Here, we have investigated whether a tyrosine kinase, PYK2, is functionally involved in AngII-induced c-Jun N-terminal kinase (JNK) activation in vascular smooth muscle cells (VSMCs). Adenovirus expressing PYK2 kinase-inactive mutant K457A or a tyrosine phosphorylation site mutant Y402F was transfected in VSMCs. AngII-induced JNK phosphorylation was markedly enhanced by K457A, whereas it was suppressed by Y402F. Protein synthesis induced by AngII was also enhanced by K457A and inhibited by Y402F. In this regard, K457A suppressed PYK2 kinase activation by AngII, whereas it enhanced AngII-induced PYK2 Tyr(402) phosphorylation. By contrast, Y402F inhibited PYK2 Tyr(402) phosphorylation, whereas it markedly enhanced AngII-induced PYK2 kinase activation. Thus, we conclude that PYK2 kinase activity negatively regulates JNK activation and protein synthesis, whereas Tyr(402) phosphorylation positively regulates these events in AngII-stimulated VSMCs, suggesting a unique role of PYK2 in mediating vascular remodeling.     

Glucagon-like peptide-1 does not mediate amylase release from AR42J cells.

In this study, AR42J pancreatic acinar cells were used to investigate if glucagon-like peptide-1 (GLP-1) or glucagon might influence amylase release and acinar cell function. We first confirmed the presence of GLP-1 receptors on AR42J cells by reverse trasncriptase-polymerase chain reaction (RT-PCR), Western blotting, and partial sequencing analysis. While cholecystokinin (CCK) increased amylase release from AR42J cells, GLP-1, alone or in the presence of CCK, had no effect on amylase release but both CCK and GLP-1 increased intracellular calcium. Similar to GLP-1, glucagon increased both cyclic adenosine monophosphate (cAMP) and intracellular calcium in AR42J cells but it actually decreased CCK-mediated amylase release (n = 20, P < 0.01). CCK stimulation resulted in an increase in tyrosine phosphorylation of several cellular proteins, unlike GLP-1 treatment, where no such increased phosphorylation was seen. Instead, GLP-1 decreased such protein phosphorylations. Genestein blocked CCK-induced phosphorylation events and amylase secretion while vanadate increased amylase secretion. These results provide evidence that tyrosine phosphorylation is necessary for amylase release and that signaling through GLP-1 receptors does not mediate amylase release in AR42J cells. J. Cell. Physiol. 181:470-478, 1999. Published 1999 Wiley-Liss, Inc.     

The Src family kinase, Lyn, is activated in pancreatic acinar cells by gastrointestinal hormones/neurotransmitters and growth factors which stimulate its association with numerous other signaling molecules

Src family kinases (SFK) play a central signaling role for growth factors, cytokines, G-protein-coupled receptors and other stimuli. SFKs play important roles in pancreatic acinar cell secretion, endocytosis, growth, cytoskeletal integrity and apoptosis, although little is known of the specific SFKs involved. In this study we demonstrate the SFK, Lyn, is present in rat pancreatic acini and investigate its activation/signaling. Ca(2+)-mobilizing agents, cAMP-mobilizing agents and pancreatic growth factors activated Lyn. CCK, a physiological regulator of pancreatic function, rapidly activated Lyn. The specific SFK inhibitor, PP2, decreased Lyn activation; however, the inactive analogue, PP3, had no effect. Inhibition of CCK-stimulated changes in [Ca(2+)](i) decreased Lyn activation by 55%; GFX, a PKC inhibitor by 36%; and the combination by 95%. CCK activation of Lyn required stimulation of high and low affinity CCK(A) receptor states. CCK stimulated an association of Lyn with PKC-delta, Shc, p125(FAK) and PYK2 as well as with their autophosphorylated forms, but not with Cbl, p85, p130(CAS) or ERK 1/2. These results show Lyn is activated by diverse pancreatic stimulants. CCK's activation of Lyn is likely an important mediator of its ability to cause tyrosine phosphorylation of numerous important cellular mediators such as p125(FAK), PYK2, PKC-delta and Shc, which play central roles in CCK's effects on acinar cell function.     

Phosphoproteomic characterization of PYK2 signaling pathways involved in osteogenesis

The PYK2 tyrosine kinase is a negative regulator of bone formation, but aside from the requirement for PYK2 kinase activity there has been little progress toward understanding of the molecular mechanism involved in this function. To gain insight into the signaling pathways modulated by PYK2 we sought to identify PYK2 substrates. Challenges inherent to a quantitative phosphoproteomic analysis for non-receptor tyrosine kinases were overcome by employing an inducible PYK2 overexpression system in NIH3T3 cells in combination with a selective PYK2 inhibitor. The identification of a number of known PYK2 substrates and interacting partners validated the methodology. Results of the inducible cell system were extended to a cell model of osteogenesis, examining the effect of the PYK2 inhibitor on the phosphorylation state of targets identified in the phosphoproteomic study. Consistent with phosphoproteomic analysis, increased osteogenesis associated with a selective PYK2 inhibitor was accompanied by reduced phosphorylation of paxillin, Gab1 and p130^Cas, along with reduction of phosphorylation levels of the Met activation loop. These results further confirmed the utility of the methodology and point to a previously unknown bi-directional activation pathway between PYK2 and Met.     

Novel tumor-promoting property of tamoxifen.

The tumor-promoting phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) cooperates with c-Src overexpression to transform rat fibroblasts. TPA transforms c-Src-overexpressing cells by depleting the delta isoform of protein kinase C (PKCdelta). Tamoxifen, which has both estrogen-mimetic and estrogen-antagonist properties, has been widely used to improve the prognosis of breast cancer patients. However, with extended use, there is an increased risk for endometrial and other cancers that can be observed within 10 years of treatment. We report here that tamoxifen, similar to TPA, cooperates with c-Src overexpression to transform 3Y1 rat fibroblasts. Tamoxifen induced both DNA synthesis and anchorage-independent cell proliferation in c-Src-overexpressing, but not in parental, 3Y1 rat fibroblasts. Tamoxifen also induced an association between c-Src and PKCdelta that resulted in the tyrosine phosphorylation and down-regulation of PKCdelta. These phenotypes were not induced by estrogen, indicating that the effect of tamoxifen was in addition to any estrogen-mimetic effects. Thus, in addition to the hyperplasia-inducing capability of an estrogen-mimetic, tamoxifen has an additional tumor-promoting capability similar to that of TPA. The dual tumor-promoting capability of both estrogen- and TPA-mimetic properties for tamoxifen may contribute to the increased incidence of endometrial cancers observed in the relatively short exposure period of <10 years.