Phosphospecific Site Tyrosine Phosphorylation of p125FAK and Proline-rich Kinase 2 Is Differentially Regulated by Cholecystokinin Receptor Type A Activation in Pancreatic Acini

Abstract

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 closely related nonreceptor focal adhesion tyrosine kinases (FAK) 1 p125 FAK and PYK2 transduce key extracellular signals that are involved in mediating growth, adhesion, cytoskeletal changes, and cellular motility (1-3). These kinases are activated by such diverse stimuli as integrins, some G protein-coupled receptors, growth factors, bioactive lipids, oncogenes (2, 4 -7), and mechanical factors (pressure, stretch, and shock) (2, 8 -10). PYK2 and FAK share 45% overall sequence homology and undergo tyrosine phosphorylation at related sites (1-3, 11). During activation FAK tyrosine phosphorylation occurs at six or more sites in vivo (11). Two sites are located within the N-terminal domain (pY397 and pY407), two sites are within the kinase activation loop (pY576 and pY577), and two sites are within the C-terminal domain (pY861 and pY925). The pY397FAK site serves as an autophosphorylation site (11) and once phosphorylated as a binding site for c-Src family protein tyrosine kinases (12, 13) and other proteins such as phosphatidylinositol 3-kinase (14) and phospholipase C␥ (15). The phosphorylation of pY576 and pY577 in the kinase activation loop is essential for full catalytic activity (11). Phosphorylation of pY925 FAK in the C-terminal domain is followed by binding of SH-2 domain containing proteins such as Grb2 (16). The role...

The p21-activated kinase, PAK2, is important in the activation of numerous pancreatic acinar cell signaling cascades and in the onset of early pancreatitis events

“…In conclusion, as summarized in Figure 12, this study demonstrates in rat pancreatic acini the p21-activated kinase, PAK2, modulates cellular cascades that have been shown to be important in mediating numerous CCK-mediated physiological and pathophysiological effects [12,12,13,15-19]. Our results demonstrate activation of PAK2 by CCK under physiological conditions plays an important role in the activation of the focal adhesion kinases (p125 FAK , PYK2); the adaptor, scaffold proteins (p130 CAS , paxillin); MAPKs (ERK,JNK, p42/44), and PI3K/Akt pathways, that are all known for mediating physiological and pathophysiological changes induced by CCK in pancreatic acinar cells.…”

“…at 37 °C as described previously [13,15]. After pre-incubation 1 ml aliquots of dispersed acini were incubated at 37 °C with or without stimulants.…”

Gastrointestinal hormones/neurotransmitters and growth factors can activate P21 activated kinase 2 in pancreatic acinar cells by novel mechanisms

“…After collagenase digestion, dispersed acini were pre-incubated in standard incubation solution for 2 hrs at 37 °C as described previously [34,39]. After pre-incubation 1 ml aliquots of dispersed acini were incubated at 37 °C with or without stimulants.…”

Cholecystokinin and Gastrin Receptors

“…Phosphorylation of p125-FAK, p130Cas, and paxillin have been reported for both CCK1R and CCK2R in numerous cell models. These phosphorylations are regulated by the small GTPase Rho that also plays an important role in stress fiber formation and modulation of cell morphology observed in response to activation of CCK receptors (163, 239,365,463,517,518,586).…”