Distinct Effects of Mitogens and the Actin Cytoskeleton on CREB and Pocket Protein Phosphorylation Control the Extent and Timing of Cyclin A Promoter Activity

Abstract

Soluble mitogens and adhesion-dependent organization of the actin cytoskeleton are required for cells to enter S phase in fibroblasts. The induction of cyclin A is also required for S-phase entry, and we now report that distinct effects of mitogens and the actin cytoskeleton on the phosphorylation of CREB and pocket proteins regulate the extent and timing of cyclin A promoter activity, respectively. First, we show that CREB phosphorylation and binding to the cyclic AMP response element (CRE) determines the extent, but not the timing, of cyclin A promoter activity. Second, we show that pocket protein inactivation regulates the timing, but not the extent, of cyclin A promoter activity. CREB phosphorylation and CRE occupancy are regulated by soluble mitogens alone, while the phosphorylation of pocket proteins requires both mitogens and the organized actin cytoskeleton. Mechanistically, cytoskeletal integrity controls pocket protein phosphorylation by allowing for sustained ERK signaling and, thereby, the expression of cyclin D1. Our results lead to a model of cyclin A gene regulation in which mitogens play a permissive role by stimulating early G₁-phase phosphorylation of CREB and a distinct regulatory role by cooperating with the organized actin cytoskeleton to regulate the duration of ERK signaling, the expression of cyclin D1, and the timing of pocket protein phosphorylation.

ACKNOWLEDGMENTS

M.E.B. and M.B. contributed equally to this study.

This work was supported by NIH grants GM48224 and CA72639 to R.K.A. M.E.B. was supported by a postdoctoral fellowship from the Department of the Army.

We thank Lou Laimins, Michael Weber, Joelle Sobczak, and Berthold Henglein for expression vectors.