Activation of cyclin-dependent kinase 4 (cdk4) by mouse MO15-associated kinase.
The assembly of functional holoenzymes composed of regulatory D-type cyclins and cyclin-dependent kinases (cdks) is rate limiting for progression through the G1 phase of the mammalian somatic cell cycle. Complexes between D-type cyclins and their major catalytic subunit, cdk4, are catalytically inactive until cyclin-bound cdk4 undergoes phosphorylation on a single threonyl residue (Thr-172). This step is catalyzed by a cdk-activating kinase (CAK) functionally analogous to the enzyme which phosphorylates cdc2 and cdk2 at Thr-161/160. Here, we demonstrate that the catalytic subunit of mouse cdc2/cdk2 CAK (a 39-kDa protein designated p39MO15) can assemble with a regulatory protein present in either insect or mammalian cells to generate a CAK activity capable of phosphorylating and enzymatically activating both cdk2 and cdk4 in complexes with their respective cyclin partners. A newly identified 37-kDa cyclin-like protein (cyclin H [R. P. Fisher and D. O. Morgan, Cell 78:713-724, 1994]) can assemble with p39MO15 to activate both cyclin A-cdk2 and cyclin D-cdk4 in vitro, implying that CAK is structurally reminiscent of cyclin-cdk complexes themselves. Antisera produced to the p39MO15 subunit can completely deplete mammalian cell lysates of CAK activity for both cyclin A-cdk2 and cyclin D-cdk4, with recovery of activity in the resulting immune complexes. By using an immune complex CAK assay, CAK activity for cyclin A-cdk2 and cyclin D-cdk4 was detected both in quiescent cells and invariantly throughout the cell cycle. Therefore, although it is essential for the enzymatic activation of cyclin-cdk complexes, CAK appears to be neither rate limiting for the emergence of cells from quiescence nor subject to upstream regulatory control by stimulatory mitogens.
Matsuoka
M
M
Howard Hughes Medical Institute, St. Jude Children's Research Hospital, Memphis, Tennessee 38105.
Kato
J Y
JY
Fisher
R P
RP
Morgan
D O
DO
Sherr
C J
CJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle; D003001:Cloning, Molecular; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D015342:DNA Probes / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014981:Xenopus; D029867:Xenopus Proteins; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D015342:DNA Probes; D018076:DNA, Complementary; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495900:CDK4 protein, human; C495891:Cdk2 protein, Xenopus; C495893:Cdk2 protein, mouse; C495902:Cdk4 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase