Distinct effects of mitogens and the actin cytoskeleton on CREB and pocketprotein phosphorylation control the extent and timing of cyclin A promoteractivity

Soluble mitogens and adhesion-dependent organization of the actin cytoskele ton are required for cells to enter S phase in fibroblasts. The induction o f cyclin A is also required for S-phase entry, and we now report that disti nct effects of mitogens and the actin cytoskeleton on the phosphorylation o f CREB and pocket proteins regulate the extent and timing of cyclin A promo ter activity, respectively. First, we show that CREB phosphorylation and bi nding to the cyclic AMP response element (CRE) determines the extent, but n ot the timing, of cyclin A promoter activity. Second, we show that pocket p rotein inactivation regulates the timing, but not the extent, of cyclin A p romoter activity. CREB phosphorylation and CRE occupancy are regulated by s oluble mitogens alone, while the phosphorylation of pocket proteins require s both mitogens and the organized actin cytoskeleton. Mechanistically, cyto skeletal integrity controls pocket protein phosphorylation by allowing for sustained ERK signaling and, thereby, the expression of cyclin D1. Our resu lts lead to a model of cyclin A gene regulation in which mitogens play a pe rmissive role by stimulating early G(1)-phase phosphorylation of CREB and a distinct regulatory role by cooperating with the organized actin cytoskele ton to regulate the duration of ERK signaling, the expression of cyclin D1, and the timing of pocket protein phosphorylation.