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.
PYK2/CAKbeta is a recently described cytoplasmic tyrosine kinase related to p125 focal adhesion kinase (p125(FAK)) that can be activated by a number of stimuli including growth factors, lipids, and some G protein-coupled receptors. Studies suggest PYK2/CAKbeta may be important for coupling various G protein-coupled receptors to the mitogen-activated protein kinase (MAPK) cascade. The hormone neurotransmitter cholecystokinin (CCK) is known to activate both phospholipase C-dependent cascades and MAPK signaling pathways; however, the relationship between these remain unclear. In rat pancreatic acini, CCK-8 (10 nM) rapidly stimulated tyrosine phosphorylation and activation of PYK2/CAKbeta by both activation of high affinity and low affinity CCK(A) receptor states. Blockage of CCK-stimulated increases in protein kinase C activity or CCK-stimulated increases in [Ca(2+)](i), inhibited by 40-50% PYK2/CAKbeta but not p125(FAK) tyrosine phosphorylation. Simultaneous blockage of both phospholipase C cascades inhibited PYK2/CAKbeta tyrosine phosphorylation completely and p125(FAK) tyrosine phosphorylation by 50%. CCK-8 stimulated a rapid increase in PYK2/CAKbeta kinase activity assessed by both an in vitro kinase assay and autophosphorylation. Total PYK2/CAKbeta under basal conditions was largely localized (77 +/- 7%) in the membrane fraction, whereas total p125(FAK) was largely localized (86 +/- 3%) in the cytosolic fraction. With CCK stimulation, both p125(FAK) and PYK2/CAKbeta translocated to the plasma membrane. Moreover CCK stimulation causes a rapid formation of both PYK2/CAKbeta-Grb2 and PYK2/CAKbeta-Crk complexes. These results demonstrate that PYK2/CAKbeta and p125(FAK) are regulated differently by CCK(A) receptor stimulation and that PYK2/CAKbeta is probably an important mediator of downstream signals by CCK-8, especially in its ability to activate the MAPK signaling pathway, which possibly mediates CCK growth effects in normal and neoplastic tissues.
Tapia
J A
JA
Departamento de Fisiología, Universidad de Extremadura, Cáceres 10071, Spain.
Ferris
H A
HA
Jensen
R T
RT
García
L J
LJ
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001692:Biological Transport; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002766:Cholecystokinin / Q000494:pharmacology*; D003078:Colchicine / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1; D051418:Focal Adhesion Kinase 2; D051416:Focal Adhesion Protein-Tyrosine Kinases; D051380:GRB2 Adaptor Protein; D066298:In Vitro Techniques; D007211:Indoles / Q000494:pharmacology; D020935:MAP Kinase Signaling System; D008297:Male; D008301:Maleimides / Q000494:pharmacology; D008954:Models, Biological; D010179:Pancreas / Q000187:drug effects*; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050737:Proto-Oncogene Proteins c-crk; D051381:Rats; D017207:Rats, Sprague-Dawley; D043886:Receptor, Cholecystokinin A; D011949:Receptors, Cholecystokinin / Q000378:metabolism*; D012844:Sincalide / Q000031:analogs & derivatives / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D015815:Cell Adhesion Molecules; C493872:Crk protein, rat; D051380:GRB2 Adaptor Protein; C495950:Grb2 protein, rat; D007211:Indoles; D008301:Maleimides; D011506:Proteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D043886:Receptor, Cholecystokinin A; D011949:Receptors, Cholecystokinin; D015638:Cytochalasin D; D000001:Calcimycin; D019284:Thapsigargin; D002766:Cholecystokinin; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051418:Focal Adhesion Kinase 2; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496052:Ptk2 protein, rat; C496054:Ptk2b protein, rat; D010738:Type C Phospholipases; C070515:bisindolylmaleimide I; D012844:Sincalide; D013755:Tetradecanoylphorbol Acetate; D003078:Colchicine; D002118:Calcium