Recent studies show that gastrointestinal hormones, similar to many growth factors, may stimulate cell growth by stimulating intracellular tyrosine phosphorylation signaling cascades. However at present little is known about the ability of these G-protein-coupled receptors to activate these cascades. During the year we have investigated the ability of the hormone/neurotransmitter, cholecystokinin (CCK) to activate these cascades. CCKA-R activation causes rapid tyrosine phosphorylation of p125FAK, paxillin, p130Cas, PYK2 and PKC-delta. PKC-delta activation is particularly important for such diverse cellular processes as modulation of transduction cascade and regulation of growth. Our recent studies show PKC delta activation is independent of changes in [Ca2+]i or PI3K but is regulated by activation of PKC-alpha. This year we investigated in detail the ability of CCK receptor activation to stimulate changes in PKC delta. CCK caused its rapid translocation to membrane and nuclear fractions, and increased its kinase activity. Tyrosine phosphorylation of PKC-delta, but not translocation, correlated with changes in kinase activity. CCK stimulated the association of PKC-delta with Src and this was essential for its activation and tyrosine phosphorylation, but not for translocation. Focal adhesion kinases (p125FAK,PYK) are important intracellular signals mediating effects of integrins, growth factors, oncogenes, bioactive lipids and some G protein-coupled receptors on growth, adhesion, cellular motility and cytoskeletal changes. Little is known on the ability of GPCR, such as the CCKA-R, to alter these kinases. We demonstrated CCK can stimulate tyrosine phosphorylation at three p125FAK sites (Y397,Y577,Y925) and PYK2 sites (pY402,Y580,Y881), however, they differ in kinetics, magnitude, participation of the different receptor states, PKC and changes in cytosolic calcium. These results show that phosphorylation of these different sites is differentially regulated and involves different intracellular mechanisms in the same cell.
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