Spy1 enhances phosphorylation and degradation of the cell cycle inhibitor p27.
The cyclin dependent kinase inhibitor (CKI) p27Kip1 binds to cyclin E/CDK2 complexes and prevents premature S-phase entry. During late G1 and throughout S-phase, p27 phosphorylation at T187 leads to its subsequent degradation, which relieves CDK2 inhibition to promote cell cycle progression. However, critical events that trigger CDK2 complexes to phosphorylate p27 remain unclear. Utilizing recombinant proteins, we demonstrate that human Speedy (Spy1) activates CDK2 to phosphorylate p27 at T187 in vitro. Addition of Spy1 or Spy1/CDK2 to a preformed, inhibited cyclin E/CDK2/p27 complex also promoted this phosphorylation. Furthermore, Spy1 protected cyclin E/CDK2 from p27 inhibition toward histone H1, in vitro. Inducible Spy1 expression in U2OS cells reduced levels of endogenous p27 and exogenous p27WT, but not a p27T187A mutant. Additionally, Spy1 expression in synchronized HeLa cells enhanced T187 phosphorylation and degradation of endogenous p27 in late G1 and throughout S-phase. Our studies provide evidence that Spy1 expression enhances CDK2-dependent p27 degradation during late G1 and throughout S-phase.
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California 92093-0367, USA.
Christopher W
CW
McAndrew
Randy F
RF
Gastwirt
April N
AN
Meyer
Lisa A
LA
Porter
Daniel J
DJ
Donoghue
D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D051357:Cyclin-Dependent Kinase 2 / Q000235:genetics / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D013941:Thymine; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; C486530:SPDYA protein, human; D050760:Cyclin-Dependent Kinase Inhibitor p27; D014443:Tyrosine; D051357:Cyclin-Dependent Kinase 2; D046988:Proteasome Endopeptidase Complex; D013941:Thymine