Regulation of cell spreading during differentiation in the muscarinic M5 receptor tumor-suppressor model
✍ Scribed by Joseph R. Spoonster; Laura Masiero; Sharon A. Savage; Julie Probst; Elise C. Kohn
- Publisher
- John Wiley and Sons
- Year
- 1997
- Tongue
- French
- Weight
- 194 KB
- Volume
- 72
- Category
- Article
- ISSN
- 0020-7136
No coin nor oath required. For personal study only.
✦ Synopsis
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.