The cyclin-dependent kinase (CDK) family member PNQALRE/CCRK supports cell proliferation but has no intrinsic CDK-activating kinase (CAK) activity.
The cyclin-dependent kinases (CDKs) that drive the eukaryotic cell cycle must be phosphorylated within the activation segment (T-loop) by a CDK-activating kinase (CAK) to achieve full activity. Although a requirement for CDK-activating phosphorylation is conserved throughout eukaryotic evolution, CAK itself has diverged between metazoans and budding yeast, and fission yeast has two CAKs, raising the possibility that additional mammalian enzymes remain to be identified. We report here the characterization of PNQALRE (also known as CCRK or p42), a member of the mammalian CDK family most similar to the cell-cycle effectors Cdk1 and Cdk2 and to the CAK, Cdk7. Although PNQALRE/CCRK was recently proposed to activate Cdk2, we show that the monomeric protein has no intrinsic CAK activity. Depletion of PNQALRE by >80% due to RNA interference (RNAi) impairs cell proliferation, but fails to arrest the cell cycle at a discrete point. Instead, both the fraction of cells with a sub-G(1) DNA content and cleavage of poly(ADP-ribose) polymerase (PARP) increase. PNQALRE knockdown did not diminish Cdk2 T-loop phosphorylation in vivo or decrease CAK activity of a cell extract. In contrast, depletion of Cdk7 by RNAi causes a proportional decrease in the ability of an extract to activate recombinant Cdk2. Our data do not support the proposed function of PNQALRE/CCRK in activating CDKs, but instead reinforce the notion of Cdk7 as the major, and to date the only, CAK in mammalian cells.
Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Lara
L
Wohlbold
Stéphane
S
Larochelle
Jack C-F
JC
Liao
Geulah
G
Livshits
Juliet
J
Singer
Kevan M
KM
Shokat
Robert P
RP
Fisher
D000595:Amino Acid Sequence; D000818:Animals; D002453:Cell Cycle; D049109:Cell Proliferation; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D005091:Exons / Q000235:genetics; D020869:Gene Expression Profiling; D006801:Humans; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013737:Testis; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D012333:RNA, Messenger; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase