Regulation of cell spreading during differentiation in the muscarinic M5 receptor tumor-suppressor model.
Activation of the muscarinic receptor in Chinese hamster ovary (CHO) cells results in a reversal of the malignant phenotype for which spreading into a bipolar, fibroblastic morphology is a marker. The process of morphologic change requires multiple events, including alterations in adhesions to substrates and cytoskeletal re-arrangement. In this report, we demonstrate the calcium-dependent involvement of p125FAK in this cellular shape change using an inhibitor of ligand-induced calcium influx, carboxyamido-triazole (CAI). p125FAK becomes tyrosine-phosphorylated after exposure to the agonist carbachol (CC), reaching maximal phosphorylation prior to initiation of cellular shape change at 1 hr into CC exposure (386 +/- 103%). Phosphorylation remained elevated through the shape change (4-12 hr). CHOm5 cell exposure to the Ca2+-mobilizing agents maitotoxin and ionomycin also resulted in p125FAK phosphorylation. Inhibition of Ca2+ influx with CAI, an inhibitor of ligand-induced Ca2+ influx, had little effect on CC-induced phosphorylation but partially inhibited ionomycin-mediated p125FAK phosphorylation. While the intracellular Ca2+ chelator BAPTA failed to prevent CC-induced p125FAK tyrosine phosphorylation, it inhibited phosphorylation due to ionomycin. CC induced Ca2+-independent binding of phosphorylated p125FAK selectively to the C-terminal SH2 domain of phosphatidylinositol-3'-kinase (PI3K). Further, CC, maitotoxin and ionomycin induced in vitro kinase activity of p125FAK for the exogenous substrate poly(Glu4Tyr1). Kinase activity stimulated by all 3 agonists was inhibited by preincubation with either CAI or BAPTA. Our results indicate that increasing intracellular Ca2+ can stimulate both p125FAK autophosphorylation and kinase activity; however, p125FAK phosphorylation in response to CC also may be induced through a Ca2+-independent pathway.
Spoonster
J R
JR
Laboratory of Pathology, National Cancer Institute, Bethesda, MD 20892, USA.
Masiero
L
L
Savage
S A
SA
Probst
J
J
Kohn
E C
EC
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002448:Cell Adhesion / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology; D006224:Cricetinae; D016147:Genes, Tumor Suppressor; D010766:Phosphorylation; D011976:Receptors, Muscarinic / Q000502:physiology*; D015398:Signal Transduction
D011976:Receptors, Muscarinic; D002118:Calcium