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© The Rockefeller University Press, 0021-9525/1999//1295 $5.00
The Journal of Cell Biology, Volume 144, Number 6, , 1999 1295-1309

Quantitative Changes in Integrin and Focal Adhesion Signaling Regulate Myoblast Cell Cycle Withdrawal

Sarita K. Sastry^*, Margot Lakonishok^*, Stanley Wu, Tho Q. Truong^*, Anna Huttenlocher^*,, Christopher E. Turner^||, and Alan F. Horwitz^*

^* Department of Cell and Structural Biology, Department of Pediatrics, and Department of Biochemistry, University of Illinois, Urbana, Illinois 61801; and ^|| Department of Anatomy and Cell Biology, State University of New York Health Science Center, Syracuse, New York 13210

We previously demonstrated contrasting roles for integrin subunits and their cytoplasmic domains in controlling cell cycle withdrawal and the onset of terminal differentiation (Sastry, S., M. Lakonishok, D. Thomas, J. Muschler, and A.F. Horwitz. 1996. J. Cell Biol. 133:169–184). Ectopic expression of the integrin 5 or 6A subunit in primary quail myoblasts either decreases or enhances the probability of cell cycle withdrawal, respectively. In this study, we addressed the mechanisms by which changes in integrin subunit ratios regulate this decision. Ectopic expression of truncated 5 or 6A indicate that the 5 cytoplasmic domain is permissive for the proliferative pathway whereas the COOH-terminal 11 amino acids of 6A cytoplasmic domain inhibit proliferation and promote differentiation. The 5 and 6A cytoplasmic domains do not appear to initiate these signals directly, but instead regulate β1 signaling. Ectopically expressed IL2R-5 or IL2R-6A have no detectable effect on the myoblast phenotype. However, ectopic expression of the β1A integrin subunit or IL2R-β1A, autonomously inhibits differentiation and maintains a proliferative state. Perturbing 5 or 6A ratios also significantly affects activation of β1 integrin signaling pathways. Ectopic 5 expression enhances expression and activation of paxillin as well as mitogen-activated protein (MAP) kinase with little effect on focal adhesion kinase (FAK). In contrast, ectopic 6A expression suppresses FAK and MAP kinase activation with a lesser effect on paxillin. Ectopic expression of wild-type and mutant forms of FAK, paxillin, and MAP/erk kinase (MEK) confirm these correlations. These data demonstrate that (a) proliferative signaling (i.e., inhibition of cell cycle withdrawal and the onset of terminal differentiation) occurs through the β1A subunit and is modulated by the subunit cytoplasmic domains; (b) perturbing subunit ratios alters paxillin expression and phosphorylation and FAK and MAP kinase activation; (c) quantitative changes in the level of adhesive signaling through integrins and focal adhesion components regulate the decision of myoblasts to withdraw from the cell cycle, in part via MAP kinase.

Key Words: MAP kinase • integrins • proliferation • FAK • paxillin

Abbreviations used in this paper: BCA, bicinchoninic acid; CA-MEK, constitutively active MEK; FAK, focal adhesion kinase; HA, hemagglutinin; MAP, mitogen-activated protein; MEK, MAP/erk kinase; UT, untransfected.

Dr. Horwitz's address after 1 July 1999 will be Department of Cell Biology, Box 439, Health Sciences Center, University of Virginia, Charlottesville, VA 22908.

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