Cholecystokinin Activates PYK2/CAKbeta by a Phospholipase C-dependent Mechanism and Its Association with the Mitogen-activated Protein Kinase Signaling Pathway in Pancreatic Acinar Cells (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1997
. Recent studies show CCK stimulates tyrosine phosphorylation TYR PHOSP of a number of proteins and evidence from the pancreas and other cellular systems suggest this could be important in mediating some of CCK's growth and secretory effects. In other tissues various neuropeptides such as bombesin can cause tyrosine phosphorylation of p125 focal adhesion Ž FAK . kinase p125 and paxillin which are important in mediating their growth effects. The purpose of the present study was to determine the effects of CCK in rat pancreatic acini on the TYR PHOSP of these latter proteins. In dispersed rat Ž . pancreatic acini, cell lysates were incubated with an anti-phosphotyrosine mAb PY20 which was immunoprecipitated and Ž .
Journal of Biological Chemistry, 2003
The focal adhesion kinases, p125 FAK and proline-rich kinase 2 (PYK2), are involved in numerous processes as adhesion, cytoskeletal changes, and growth. These kinases have 45% homology and share three tyrosine phosphorylation (TyrP) sites. Little information exists on the ability of stimulants to cause TyrP of each kinase site and the cellular mechanism involved. We explored the ability of the neurotransmitter/hormone, CCK, to stimulate TyrP at each site. In rat pancreatic acini, CCK stimulated TyrP at each site in both kinases. TyrP was rapid except for pY397FAK. The magnitude of TyrP differed with the different FAK and PYK2 sites. The CCK dose-response curve for TyrP for sites in each kinase was similar. CCK-JMV, an agonist of the high affinity receptor state and antagonist of the low affinity receptor state, was less efficacious than CCK at each FAK/ PYK2 site and inhibited CCK maximal stimulation. Thapsigargin decreased CCK-stimulated TyrP of pY402PYK2 and pY925FAK but not the other sites. GF109203X reduced TyrP of only the PYK2 sites, pY402 and pY580. GF109203X with thapsigargin decreased TyrP of pY402PYK2 and the three FAK sites more than either inhibitor alone. Basal TyrP of pY397FAK was greater than other sites. These results demonstrate that CCK stimulates tyrosine phosphorylation of each of the three homologous phosphorylation sites in FAK and PYK2. However, CCK-stimulated TyrP at these sites differs in kinetics, magnitude, and participation of the high/low affinity receptor states and by protein kinase C and [Ca 2؉ ] i . These results show that phosphorylation of these different sites is differentially regulated and involves different intracellular mechanisms in the same cell. . The abbreviations used are: FAK, p125 FAK (p125 FAK focal adhesion kinase); PYK2, proline-rich kinase 2 (cell adhesion kinase ); CCK, cholecystokinin; CCK A receptor, cholecystokinin receptor type A; PKC, protein kinase C; CCK-JMV, Boc-CCK(27-32) [Nle 28,31 ]-2-phenylethylester; PLC, phospholipase C; pAb, polyclonal antibody; mAb, monoclonal antibody; HUVEC, human umbilical vein endothelial cells.
FEBS Letters, 1999
The role of the Ca 2+ -activated tyrosine kinase, Pyk2, in the pleiotropic coupling of nerve cell stimulation to the MAP kinase cascade still remains undefined. Using a panel of PC12 clones, one of which was defective in Pyk2, we demonstrate (1) that the MAP kinase response induced by a [Ca 2+ ] i rise (following application of the Ca 2+ ionophore, ionomycin) is inappreciable in the defective clone and is re-established after Pyk2 transfection; and (2) that the responses to both protein kinase C and P 2y2 receptor activation occur normally even in the defective cells. We conclude that Pyk2 is the key mediator in the pathway activated by Ca 2+ but has minor roles with the other types of stimulation.
Journal of Biological Chemistry, 1996
Many G protein-coupled receptors (e.g. that of angiotensin II) activate phospholipase C, initially increasing intracellular calcium and activating protein kinase C. In the WB and GN4 rat liver epithelial cell lines, agonistinduced calcium signals also stimulate tyrosine phosphorylation and subsequently increase the activity of c-Jun N-terminal kinase (JNK). We have now purified the major calcium-dependent tyrosine kinase (CADTK), and by peptide and nucleic acid sequencing identified it as a rat homologue of human PYK2. CADTK/PYK2 is most closely related to p125 FAK and both enzymes are expressed in WB and GN4 cells. Angiotensin II, which only slightly increases p125 FAK tyrosine phosphorylation in GN4 cells, substantially increased CADTK tyrosine autophosphorylation and kinase activity. Agonists for other G protein-coupled receptors (e.g. LPA), or those increasing intracellular calcium (thapsigargin), also stimulated CADTK. In comparing the two rat liver cell lines, GN4 cells exhibited ϳ 5-fold greater angiotensin II-and thapsigargin-dependent CADTK activation thanWBcells.AlthoughmaximalJNKactivationbystressdependent pathways (e.g. UV and anisomycin) was equivalent in the two cell lines, calcium-dependent JNK activation was 5-fold greater in GN4, correlating with CADTK activation. In contrast to JNK, the thapsigargin-dependent calcium signal did not activate mitogen-activated protein kinase and Ang II-dependent mitogen-activated protein kinase activation was not correlated with CADTK activation. Finally, while some stress-dependent activators of the JNK pathway (NaCl and sorbitol) stimulated CADTK, others (anisomycin, UV, and TNF␣) did not. In summary, cells expressing CADTK/PYK2 appear to have two alternative JNK activation pathways: one stressactivated and the other calcium-dependent.
Circulation Research, 1998
Angiotensin II (Ang II) induces vascular smooth muscle cell (VSMC) growth by activating G q-protein-coupled AT 1 receptors, which leads to elevation of cytosolic Ca 2ϩ ([Ca 2ϩ ] i) and activation of protein kinase C (PKC) and mitogen-activated protein kinases. To assess the link between these Ang II-induced signaling events, we examined the effect of Ang II on the proline-rich tyrosine kinase (PYK2), previously found to be activated by a variety of stimuli that increase [Ca 2ϩ ] i or activate PKC. PYK2 distribution was demonstrated in rat aortic tissue and in cultured VSMC by immunohistochemistry, revealing a cytosolic distribution distinct from smooth muscle ␣-actin, focal adhesion kinase, or paxillin. The involvement of PYK2 in Ang II signaling was measured by immunoprecipitation and immune complex kinase assays. Treatment of quiescent VSMC with Ang II resulted in a concentration-and time-dependent increase in PYK2 tyrosine phosphorylation and kinase activity in PYK2 immunoprecipitates. PYK2 phosphorylation was inhibited by AT 1 receptor blockade and was attenuated by downregulation of PKC or the chelation of [Ca 2ϩ ] i. Treatment with either phorbol ester or Ca 2ϩ ionophore also increased PYK2 phosphorylation, suggesting that PKC activation and/or increased [Ca 2ϩ ] i are both necessary and sufficient to activate PYK2. Activation of PYK2 by Ang II was also associated with increased PYK2-src complex formation, suggesting that PYK2 activation represents a potential link between Ang II-stimulated [Ca 2ϩ ] i and PKC activation with downstream signaling events such as mitogen-activated protein kinase activation involved in the regulation of VSMC growth.
Biological Chemistry, 2000
Recently, the involvement of the MAP kinase ERK in mitogenic signaling of cholecystokininB (CCKB) receptors has been shown. However, the intracellular effector systems involved in this signaling pathway are poorly defined. In this study, we used COS-7 cells transiently transfected with the human CCKB receptor to investigate cholecystokinin-induced MAP kinase activation. CCK-8 induced activation of ERK2 which is associated with its phosphorylation and localization in the nucleus. The CCK-8-dependent ERK stimulation is sensitive to wortmannin an inhibitor of phosphoinositide 3-kinases (PI3Ks) indicating the involvement of PI3K activity. To identify the PI3K species involved in mitogenic signaling of the CCKB receptor several dominant-negative mutants of PI3K regulatory and catalytic subunits were transiently expressed. Surprisingly, different catalytically inactive mutants of the G protein-sensitive PI3Kγ did not affect ERK stimulation induced by CCK, whereas a dominant-negative mutan...
Cancer Research
Castrili, cholecystokinin (CCK), and CCK-related peptides comprise a Imimonili family characterized by an identical carboxy-terminal amino acid sequence, a domain critical for receptor binding. The addition of gastrin to small cell lung cancer (SCLC) cells causes a rapid and transient increase in the intracellular concentration of calcium i|(';r'|,i. Further more, gastrin acts as a direct growth factor through CCKn/gastrin recep tors. We report here that the expression of the mRNA coding for CCKu/ gastrin receptors correlates with the responsiveness of SCLC cells to gastrin in terms of (';r ' mobilization and stimulation of clonal growth in semisolid medium. The GLC19 SCLC cell line had no detectable expres sion of CCKn/gastrin receptor mRNA. Accordingly, gastrin (1-100 IIMIdid not cause any measurable increase in |('¡i '' |¡-In contrast, the addition of cholecystokinin residues 26â€"33(CCK-8) caused a rapid and transient increase in |< '¡r'1 ], in this cell line. CCK-8 mobilized « ;i '' in a dosedependent manner in the nanomolar range (half-maximal stimulatory concentration = 12 nvi). Furthermore, the selective CCKA antagonist CAM-1481 inhibited the increase in |(':i-'-|, induced by CCK-8 (halfmaximal inhibitory concentration = 3 n>i) in GLC19 but not in H510 cells. The selective CCKn/gastrin antagonist blocked the increase in |(;r ' |, induced by CCK-8 (half-maximal inhibitory concentration = 80 pM) in H510 but not in GLC19 cells. Thus, the effects of CCK-8 are mediated through CCKA receptors in GLC19 cells and via CCKB/gastrin receptors in H510 cells. CCK-8 markedly stimulated colony formation in GLC19 cells in a dose-dependent manner in the nanomolar range, whereas over the same concentration range, gastrin had no effect on clonal growth. CAM-1481 inhibited the CCK-stimulated colony formation in GLC19 but not in H510 cells. Our results show, for the first time, that CCKA receptors can mediate (a'' mobilization and growth in SCLC cells and that SCLC cells express two distinct functional CCK receptor subtypes.
Biochimica et biophysica acta, 2002
PKC-delta is important in cell growth, apoptosis, and secretion. Recent studies show its stability is regulated by tyrosine phosphorylation (TYR-P), which can be stimulated by a number of agents. Many of these stimuli also activate phospholipase C (PLC) cascades and little is known about the relationship between these cascades and PKC-delta TYR-P. Cholecystokinin (CCK) stimulates PKCs but it is unknown if it causes PKC-delta TYR-P and if so, the relationship between these cascades is unknown. In rat pancreatic acini, CCK-8 stimulated rapid PKC-delta TYR-P by activation of the low affinity CCK(A) receptor state. TPA had a similar effect. BAPTA did not decrease CCK-stimulated PKC-delta TYR-P but instead, increased it. A23187 did not stimulate PKC-delta TYR-P. Wortmannin and LY 294002 did not alter CCK-stimulated PKC-delta TYR-P. GF 109203X, at low concentrations, increased PKC-delta TYR-P stimulated by CCK or TPA and at higher concentrations, inhibited it. The cPKC inhibitors, Gö 6976...
Journal of Biological Chemistry, 1998
Pyk2 is a recently described cytoplasmic tyrosine kinase that is related to focal adhesion kinase (FAK) and can be activated by a variety of stimuli that elevate intracellular calcium. In this report, we showed that Pyk2 and FAK tyrosine phosphorylation are regulated differentially by integrin-mediated cell adhesion and soluble factors both in rat aortic smooth muscle cells, which express endogenous Pyk2 and FAK, and in transfected Chinese hamster ovary cells. We also found that Pyk2 is diffusely present throughout the cytoplasm, while FAK is localized in focal contacts as expected, suggesting that the different localization may account for their differential regulation. By analyzing a chimeric protein contain N-terminal and kinase domains of Pyk2 and C-terminal domain of FAK, we provided evidence that the distinctive C-terminal domains of Pyk2 and FAK were responsible for their differential regulation by integrins and soluble stimuli as well as their subcellular localization. Finally, we correlated FAK, Pyk2, and the chimeric protein binding to talin, but not paxillin, with their regulation by integrins and focal contact localization. These results demonstrate that the distinctive C-terminal domain of Pyk2 and FAK confer their differential regulation by different subcellular localization and association with the cytoskeletal protein talin.
Journal of Biological Chemistry, 1999
The stress-activated p38 mitogen-activated protein kinase (p38 MAPK), a member of the subgroup of mammalian kinases, appears to play an important role in regulating inflammatory responses, including cytokine secretion and apoptosis. The upstream mediators that link extracellular signals with the p38 MAPK signaling pathway are currently unknown. Here we demonstrate that pp125 focal adhesion kinase-related tyrosine kinase RAFTK (also known as PYK2, CADTK) is activated specifically by methylmethane sulfonate (MMS) and hyperosmolarity but not by ultraviolet radiation, ionizing radiation, or cis-platinum. Overexpression of RAFTK leads to the activation of p38 MAPK. Furthermore, overexpression of a dominant-negative mutant of RAFTK (RAFTK K-M) inhibits MMS-induced p38 MAPK activation. MKK3 and MKK6 are known potential constituents of p38 MAPK signaling pathway, whereas SEK1 and MEK1 are upstream activators of SAPK/JNK and ERK pathways, respectively. We observe that the dominantnegative mutant of MKK3 but not of MKK6, SEK1, or MEK1 inhibits RAFTK-induced p38 MAPK activity. Furthermore, the results demonstrate that treatment of cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid, tetra(acetoxymethyl)-ester, a membranepermeable calcium chelator, inhibits MMS-induced activation of RAFTK and p38 MAPK. Taken together, these findings indicate that RAFTK represents a stress-sensitive mediator of the p38 MAPK signaling pathway in response to certain cytotoxic agents. The mitogen-activated protein kinases (MAPKs) 1 are induced in response to diverse classes of inducers in the trans-* This investigation was supported by Public Health Service Grants CA75216 (to S. K.), CA65861 (to R. J. D.) awarded by NCI, and by RO1 HL55445 (to S. A.