Phosphorylation of RIalpha by cyclin-dependent kinase CDK 2/cyclin E modulates the dissociation of the RIalpha-RFC40 complex.
We have previously demonstrated that the nuclear transport of the second subunit of the Replication Factor C complex, RFC40, by the regulatory subunit, RIalpha, of PKA is cell cycle specific and impairment in this transport results in G(1) arrest. In this study, we have investigated whether the cyclin-dependent kinases play a role in regulating the RIalpha-RFC40 complex formation. In this context, we have identified RIalpha as a novel substrate for the G(1)/S-Cyclin-dependent kinase, CDK2/Cyclin E, and found that RIalpha is specifically phosphorylated at the serine residue. Treatment of MCF7 cells with a CDK inhibitor, olomoucine, resulted in a significant accumulation in the RIalpha-RFC40 complex by 3.10 +/- 0.08 fold and a parallel decrease in the RFC40-37 complex formation by 73.73 +/- 11.81%. Furthermore, in vitro phosphorylation experiments suggest that, phosphorylation of RIalpha by CDK2/CyclinE kinase promotes the dissociation of the RIalpha-RFC40 complex and that once RIalpha is phosphorylated it cannot complex with RFC40. Inhibition of the serine-threonine phosphatase, PP1, by Calyculin A, significantly reduced the RIalpha-RFC40 complex formation, substantiating the in vitro phosphorylation data. Taken together, these findings suggest that CDK2/Cyclin E may function as downstream modulator that regulates the dissociation of the RIalpha-RFC40 complex and subsequently the association of the RFC40-RFC37 complex.
Gupte
Rakhee S
RS
Department of Biochemistry & Molecular Biology, New York Medical College, Valhalla, New York 10595, USA. rakhee_gupte@nymc.edu
Traganos
Frank
F
Darzynkiewicz
Zbigniew
Z
Lee
Marietta Y W T
MY
D021581:Active Transport, Cell Nucleus / Q000235:genetics; D045744:Cell Line, Tumor; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D019927:Cyclin E / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D051818:Replication Protein C / Q000378:metabolism*
D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D019927:Cyclin E; D004791:Enzyme Inhibitors; C517834:PRKAR1A protein, human; C497561:RFC2 protein, human; D017868:Cyclic AMP-Dependent Protein Kinases; D051357:Cyclin-Dependent Kinase 2; D051818:Replication Protein C