Spy1 interacts with p27Kip1 to allow G1/S progression.
Progression through the G1/S transition commits cells to synthesize DNA. Cyclin dependent kinase 2 (CDK2) is the major kinase that allows progression through G1/S phase and subsequent replication events. p27 is a CDK inhibitor (CKI) that binds to CDK2 to prevent premature activation of this kinase. Speedy (Spy1), a novel cell cycle regulatory protein, has been found to prematurely activate CDK2 when microinjected into Xenopus oocytes and when expressed in mammalian cells. To determine the mechanism underlying Spy1-induced proliferation in mammalian cell cycle regulation, we used human Spy1 as bait in a yeast two-hybrid screen to identify interacting proteins. One of the proteins isolated was p27; this novel interaction was confirmed both in vitro, using bacterially expressed and in vitro translated proteins, and in vivo, through the examination of endogenous and transfected proteins in mammalian cells. We demonstrate that Spy1 expression can overcome a p27-induced cell cycle arrest to allow for DNA synthesis and CDK2 histone H1 kinase activity. In addition, we utilized p27-null cells to demonstrate that the proliferative effect of Spy1 depends on the presence of endogenous p27. Our data suggest that Spy1 associates with p27 to promote cell cycle progression through the G1/S transition.
Porter
Lisa A
LA
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California 92093-0367, USA.
Kong-Beltran
Monica
M
Donoghue
Daniel J
DJ
D016475:3T3 Cells; D000818:Animals; D042846:CDC2-CDC28 Kinases / Q000096:biosynthesis / Q000378:metabolism*; D019556:COS Cells; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D051357:Cyclin-Dependent Kinase 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004789:Enzyme Activation; D004794:Enzyme Repression; D016193:G1 Phase; D051379:Mice; D018345:Mice, Knockout; D011485:Protein Binding / Q000502:physiology; D016196:S Phase; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; C486530:SPDYA protein, human; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D042846:CDC2-CDC28 Kinases; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2