ERK1/2 and p38 cooperate to induce a p21 super(CIP1)-dependent G sub(1) cell cycle arrest

To study the mechanisms by which mitogen- and stress-activated protein kinases regulate cell cycle re-entry, we have used a panel of conditional kinases that stimulate defined MAPK or SAPK cascades. Activation of Delta MEKK3:ER* during serum restimulation of quiescent cells causes a strong activation of JNK1 and p38 alpha but only a modest potentiation of serum-stimulated ERK1/2 activity. In CCl39 cells this promoted a sustained G sub(1) arrest that correlated with decreased expression of cyclin D1 and Cdc25A, increased expression of p21 super(CIP1) and inhibition of CDK2 activity. In Rat-1 cells, in which p21 super(CIP1) expression is silenced by methylation, Delta MEKK3:ER* activation caused only a transient delay in the S phase entry rather than a sustained G sub(1) arrest. Furthermore, p21 super(CIP1-/-) 3T3 cells were defective for the Delta MEKK3:ER*-induced G sub(1) cell cycle arrest compared to their wild-type counterparts. These results suggest that activated Delta MEKK3:ER* inhibits the G sub(1) arrow right S progression by two kinetically distinct mechanisms, with expression of p21 super(CIP1) being required to ensure a sustained G sub(1) cell cycle arrest. The ERK1/2 and p38 alpha beta pathways cooperated to induce p21 super(CIP1) expression and inhibition of p38 alpha beta caused a partial reversal of the cell cycle arrest. In contrast, selective activation of ERK1/2 by Delta Raf-1:ER* did not inhibit serum stimulated cell cycle re-entry. Finally, selective activation of JNK by Delta MEKK1:ER* failed to inhibit cell cycle re-entry, even in cells that retained wild-type p53, arguing against a major role for JNK alone in antagonizing the G sub(1) arrow right S transition.