A Discrete Cell Cycle Checkpoint in Late G1 That Is Cytoskeleton-Dependent and MAP Kinase (Erk)-Independent

Abstract

Cell spreading on extracellular matrix and associated changes in the actin cytoskeleton (CSK) are necessary for progression through G1 and entry into S phase of the cell cycle. Pharmacological disruption of CSK integrity inhibits early mitogenic signaling to the extracellular signal-regulated kinase (Erk) subfamily of the mitogen-activated protein kinases (MAPKs) and arrests the cell cycle in G1. Here we show that this block of G1 progression is not simply a consequence of inhibition of the MAPK/Erk pathway but instead it reveals the existence of a discrete CSK-sensitive checkpoint. Use of PD98059 to inhibit MAPK/Erk and cytochalasin D (Cyto D) to disrupt the actin CSK at progressive time points in G1 revealed that the requirement for MAPK/Erk activation lasts only to mid-G1, while the actin CSK must remain intact up to late G1 restriction point, R, in order for capillary endothelial cells to enter S phase. Additional analysis using Cyto D pulses defined a narrow time window of 3 h just prior to R in which CSK integrity was shown to be critical for the G1/S transition. Cyto D treatment led to down-regulation of cyclin D1 protein and accumulation of the cdk inhibitor, p27Kip1, independent of cell cycle phase, suggesting that these changes resulted directly from CSK disruption rather than from a general cell cycle block. Together, these data indicate the existence of a distinct time window in late G1 in which signals elicited by the CSK act independently of early MAPK/Erk signals to drive the cell cycle machinery through the G1/S boundary and, hence, promote cell growth.