Autocatalytic phosphorylation of CDK2 at the activating Thr160.
Phosphorylation of a critical residue in the activation loop of many protein kinases is essential for enzymatic activity. The trimeric complex CDK7/cyclin H/Mat1 phosphorylates the cell cycle regulated cyclin-dependent kinase, CDK2 at Thr-160 in the activation segment in vitro. Whether CDK7/cyclin H is the in vivo CDK2 activating kinase (CAK), or the sole CAK for CDK2 remains elusive. Here we show that monomeric human CDK2 purified from bacteria is phosphorylated at Thr-160. CDK2 expressed and purified from bacteria exhibited kinase activity against histone H1, which was stimulated by cyclin E or A expressed and purified from bacteria. The kinase activity was dependent on both the catalytic activity of CDK2 and Thr-160 phosphorylation since it was abolished when CDK2 was mutated at Lys33 in the ATP binding site (K33R) or Thr160 (T160A) or when treated with lambda phosphatase. Mass spectrometry based phosphopeptide mapping confirmed the phosphorylation of bacterial CDK2 on Thr160. Consistent with a role of CDK2 in auto-activation, inhibition of CDK2 in human cells either by pharmacological inhibition of CDK2 or by the coexpression of the CDK2 inhibitors p21 or p27, inhibited CDK2 Thr-160 phosphorylation. Our results demonstrate that CDK2 is capable of autophosphorylation at Thr160.
Abbas
Tarek
T
Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
Jha
Sudhakar
S
Sherman
Nicholas E
NE
Dutta
Anindya
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D020134:Catalytic Domain / Q000502:physiology*; D002460:Cell Line; D045744:Cell Line, Tumor; D051357:Cyclin-Dependent Kinase 2 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006657:Histones / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D013912:Threonine / Q000737:chemistry / Q000378:metabolism*
D050759:Cyclin-Dependent Kinase Inhibitor p21; D004791:Enzyme Inhibitors; D006657:Histones; D050760:Cyclin-Dependent Kinase Inhibitor p27; D013912:Threonine; D000255:Adenosine Triphosphate; D011494:Protein Kinases; C102333:histone H1 kinase; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2