CCK causes rapid tyrosine phosphorylation of p125(FAK) focal adhesion kinase and paxillin in rat pancreatic acini

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 kinase (p125(FAK)) 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 pancreatic acini, cell lysates were incubated with an anti-phosphotyrosine mAb (PY20) which was immunoprecipitated and then analyzed by Western blotting with anti phosphotyrosine mAb (4G10), anti-p125(FAK) mAb or anti-paxillin mAb. CCK-8 at 5 min increased TYR PHOSP of five proteins of molecular weight > 60 000 including a broad M-r 110-130 000 and M-r 70-80 000, An increase in TYR PHOSP of both p125(FAK) and paxillin was detected within 1 min of adding CCK and reached a maximum at 2.5 min with a 9.1 +/- 1.9-fold increase for p125(FAK) and 3.6 +/- 0.6-fold for paxillin. CCK-8 caused a half-maximal increase in TYR PHOSP of p125(FAK) at 0.1 nM and paxillin at 0.03 nM. CCK-JMV also stimulated an increase in TYR PHOSP of both proteins, but was only 50% as efficacious as CCK-8, CCK-JMV caused a half-maximal increase at 10 nM and maximal at 1 mu M for both proteins. To investigate whether the low affinity CCK receptor state also caused TYR PHOSP of both proteins, increasing concentrations of CCK-JMV were added to a maximally effective CCK-8 concentration (1 nM). Detectable inhibition of CCK-8-stimulated TYR PHOSP occurred with 1 mu M CCK-JMV and with 3 mu M CCK-JMV the CCK-8-stimulated response was inhibited 50% and was the same as that seen with CCK-JMV alone. These studies demonstrate that in rat pancreatic acini, CCK causes rapid TYR PHOSP of both p125(FAK) and paxillin. This stimulation is mediated by both the high affinity and low affinity CCK receptor states. This phosphorylation of these proteins could be important in mediating CCK's effect on the cytoskeleton or growth effects as shown for a number of other agents (oncogenes, neuropeptides, integrins). (C) 1997 Elsevier Science B.V.