Regulated activating Thr172 phosphorylation of cyclin-dependent kinase 4(CDK4): its relationship with cyclins and CDK "inhibitors".
Cyclin-dependent kinase 4 (CDK4) is a master integrator of mitogenic and antimitogenic extracellular signals. It is also crucial for many oncogenic transformation processes. Various molecular features of CDK4 activation remain poorly known or debated, including the regulation of its association with D-type cyclins, its activating Thr172 phosphorylation, and the roles of Cip/Kip CDK "inhibitors" in these processes. Thr172 phosphorylation of CDK4 was reinvestigated using two-dimensional gel electrophoresis in various experimental systems, including human fibroblasts, canine thyroid epithelial cells stimulated by thyrotropin, and transfected mammalian and insect cells. Thr172 phosphorylation of CDK4 depended on prior D-type cyclin binding, but Thr172 phosphorylation was also found in p16-bound CDK4. Opposite effects of p27 on cyclin D3-CDK4 activity observed in different systems depended on its stoichiometry in this complex. Thr172-phosphorylated CDK4 was enriched in complexes containing p21 or p27, even at inhibitory levels of p27 that precluded CDK4 activity. Deletion of the p27 nuclear localization signal sequence relocalized cyclin D3-CDK4 in the cytoplasm but did not affect CDK4 phosphorylation. Within cyclin D3 complexes, T-loop phosphorylation of CDK4, but not of CDK6, was directly regulated, identifying it as a determining target for cell cycle control by extracellular factors. Collectively, these unexpected observations indicate that CDK4-activating kinase(s) should be reconsidered.
Bockstaele
Laurence
L
Institute of Interdisciplinary Research, Université Libre de Bruxelles, Campus Erasme, B-1070 Brussels, Belgium.
Kooken
Hugues
H
Libert
Frederick
F
Paternot
Sabine
S
Dumont
Jacques E
JE
de Launoit
Yvan
Y
Roger
Pierre P
PP
Coulonval
Katia
K
D000818:Animals; D002478:Cells, Cultured; D051358:Cyclin-Dependent Kinase 4 / Q000032:analysis / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism*; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology; D004285:Dogs; D004789:Enzyme Activation; D006801:Humans; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D019913:Nuclear Localization Signals; D050760:Cyclin-Dependent Kinase Inhibitor p27; D013912:Threonine; D012694:Serine; D020794:Receptor Protein-Tyrosine Kinases; D051358:Cyclin-Dependent Kinase 4