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 selective disruption of the actin cytoskeleton by dihydrocytochalasin B (H(2)CB) blocked the mitogenic effect in normal Swiss 3T3 cells, leading to cell cycle arrest at mid to late G(1) phase. Cells treated with H(2)CB remain tightly attached to the substratum and respond to mitogen-induced MAP kinase activation. Upon cytoskeleton disruption, however, growth factors fail to induce hyperphosphorylation of the retinoblastoma protein (pRb) and the pRb-related p107. While cyclin D1 induction and cdk4-associated kinase activity are not affected, induction of cyclin E expression and activation of cyclin E-cdk2 complexes are greatly inhibited in growth-stimulated cells treated with H(2)CB. The inhibition of cyclin E expression appears to be mediated at least in part at the RNA level and the inhibition of cdk2 kinase activity is also attributed to the decrease in cdk2 phosphorylation and proper subcellular localization. The expression patterns of cdk inhibitors p21 and p27 are similar in both untreated and H(2)CB-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 structure affects nuclear migration of p107 during G(1)-to-S progression. Taken together, our results suggest that the actin cytoskeleton-dependent G(1) arrest is linked to the cyclin-cdk pathway. We hypothesize that normal actin structure may be important for proper localization of certain G(1) regulators, consequently modulating specific cyclin and kinase expression.
Reshetnikova
G
G
Department of Pediatrics, Children's Hospital and The Ohio State University College of Medicine and Public Health, Columbus, Ohio, 43205, USA.
Barkan
R
R
Popov
B
B
Nikolsky
N
N
Chang
L S
LS
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001798:Blood Proteins / Q000494:pharmacology; D015153:Blotting, Western; D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins; D002467:Cell Nucleus / Q000737:chemistry / Q000201:enzymology; D019938:Cyclin D1 / Q000032:analysis / Q000378:metabolism; D019927:Cyclin E / Q000032:analysis / Q000235:genetics*; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000032:analysis / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism; D003571:Cytochalasin B / Q000031:analogs & derivatives / Q000494:pharmacology; D003593:Cytoplasm / Q000737:chemistry / Q000201:enzymology; D003599:Cytoskeleton / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D016193:G1 Phase / Q000187:drug effects / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008934:Mitogens / Q000494:pharmacology; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000032:analysis; D016160:Retinoblastoma Protein / Q000378:metabolism; D050720:Retinoblastoma-Like Protein p107; D016196:S Phase / Q000187:drug effects / Q000502:physiology; D025521:Tumor Suppressor Proteins
D000199:Actins; D001798:Blood Proteins; C493957:Cdkn1a protein, mouse; C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D019927:Cyclin E; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D008869:Microtubule-Associated Proteins; D008934:Mitogens; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; C489485:Rbl1 protein, mouse; D016160:Retinoblastoma Protein; D050720:Retinoblastoma-Like Protein p107; D025521:Tumor Suppressor Proteins; D019938:Cyclin D1; D050760:Cyclin-Dependent Kinase Inhibitor p27; C024794:dihydrocytochalasin B; D003571:Cytochalasin B; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495893:Cdk2 protein, mouse; C495902:Cdk4 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases