Disruption of the actin cytoskeleton leads to inhibition of mitogen-induced cyclin E expression, cdk2 phosphorylation, and nuclear accumulation of the retinoblastoma protein-related p107 protein

The actin cytoskeleton has been found to be required for mitogen-stimulated cells to passage through the cell cycle checkpoint. Here we show that sele ctive disruption of the actin cytoskeleton by dihydrocytochalasin B (H2CB) blocked the mitogenic effect in normal Swiss 3T3 cells, leading to cell cyc le arrest at mid to late G(1) phase. Cells treated with H2CB remain tightly attached to the substratum and respond to mitogen-induced MAP kinase activ ation. Upon cytoskeleton disruption, however, growth factors fail to induce hyperphosphorylation of the retinoblastoma protein (pRb) and the pRb-relat ed p107. While cyclin D1 induction and cdk4-associated kinase activity are not affected, induction of cyclin E expression and activation of cyclin E-c dk2 complexes are greatly inhibited in growth-stimulated cells treated with H2CB. The inhibition of cyclin E expression appears to be mediated at leas t in part at the RNA level and the inhibition of cdk2 kinase activity is al so attributed to the decrease in cdk2 phosphorylation and proper subcellula r localization. The expression patterns of cdk inhibitors p21 and p27 are s imilar in both untreated and H2CB-treated cells upon serum stimulation. In addition, the changes in subcellular localization of pRb and p107 appear to be linked to their phosphorylation states and disruption of normal actin s tructure affects nuclear migration of p107 during G(1)-to-S progression. Ta ken together, our results suggest that the actin cytoskeleton-dependent G(1 ) arrest is linked to the cyclin-cdk pathway. We hypothesize that normal ac tin structure may be important for proper localization of certain G(1) regu lators, consequently modulating specific cyclin and kinase expression. (C) 2000 Academic Press.