A discrete cell cycle checkpoint in late G(1) that is cytoskeleton-dependent and MAP kinase (Erk)-independent.
Cell spreading on extracellular matrix and associated changes in the actin cytoskeleton (CSK) are necessary for progression through G(1) 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 G(1). Here we show that this block of G(1) 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 G(1) revealed that the requirement for MAPK/Erk activation lasts only to mid-G(1), while the actin CSK must remain intact up to late G(1) 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 G(1)/S transition. Cyto D treatment led to down-regulation of cyclin D1 protein and accumulation of the cdk inhibitor, p27(Kip1), 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 G(1) in which signals elicited by the CSK act independently of early MAPK/Erk signals to drive the cell cycle machinery through the G(1)/S boundary and, hence, promote cell growth.
Huang
Sui
S
Department of Surgery, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Ingber
Donald E
DE
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000502:physiology*; D002196:Capillaries / Q000166:cytology / Q000378:metabolism; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27; D015638:Cytochalasin D / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D016193:G1 Phase; D006801:Humans; D007700:Kinetics; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D016160:Retinoblastoma Protein / Q000378:metabolism; D016196:S Phase; D025521:Tumor Suppressor Proteins / Q000378:metabolism
D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D016160:Retinoblastoma Protein; D025521:Tumor Suppressor Proteins; D019938:Cyclin D1; D050760:Cyclin-Dependent Kinase Inhibitor p27; D015638:Cytochalasin D; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one