Regulation of cell spreading during differentiation in the muscarinic M5 receptor tumor-suppressor model.

Abstract

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.

• Authors: J R Spoonster, L Masiero, S A Savage, J Probst, E C Kohn
• Journal: International journal of cancer (Int J Cancer) Vol. 72 Issue 2 Pg. 362-8 (Jul 17 1997) ISSN: 0020-7136 [Print] United States
• PMID: 9219847 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Receptors, Muscarinic
• Calcium

Topics

• Animals
• CHO Cells
• Calcium (metabolism)
• Cell Adhesion (physiology)
• Cell Differentiation (physiology)
• Cricetinae
• Genes, Tumor Suppressor
• Phosphorylation
• Receptors, Muscarinic (physiology)
• Signal Transduction