Is Cdk7/cyclin H/MAT1 the genuine cdk activating kinase in cycling Xenopus egg extracts?
Formation of active cdk (cyclin dependent kinase)/ cyclin kinases involves phosphorylation of a conserved threonine residue in the T loop of the cdk catalytic-subunit by CAK (Cdk Activating Kinase). CAK was first purified biochemically from higher eukaryotes and identified as a trimeric complex containing a cdk7 catalytic subunit, cyclin H and MAT1 (Ménage à trois), a member of the RING finger family. The same trimeric complex is also part of basal transcription factor TFIIH. In budding yeast, the closest homologs of cdk7 and cyclin H, KIN28 and CCL1, respectively, also associate with TFIIH. However, the KIN28/CCL1 complex does not display CAK activity and a distinct protein kinase able to phosphorylate monomeric CDC28 and GST-cdk2 was recently identified, challenging the identification of cdk7 as the physiological CAK in higher eukaryotes. Here we demonstrate that immunodepletion of cdk7 suppresses CAK activity from cycling Xenopus egg extracts, and arrest them before M-phase. We also show that specific translation of mRNAs encoding Xenopus cdk7 and its associated subunits restores CAK activity in cdk7-immunodepleted Xenopus egg extracts. Hence, the cdk7 complex is necessary and sufficient for activation of cdk-cyclin complexes in cycling Xenopus egg extracts.
Fesquet
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Centre de Recherches de Biochimie Macromoléculaire, CNRS ERS 155, Montpellier, France.
Morin
N
N
Doree
M
M
Devault
A
A
D000818:Animals; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle / Q000502:physiology*; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D019925:Cyclin A / Q000235:genetics / Q000378:metabolism; D056748:Cyclin H; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D008297:Male; D008938:Mitosis; D010766:Phosphorylation; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013094:Spermatozoa / Q000737:chemistry; D014981:Xenopus / Q000196:embryology*; D029867:Xenopus Proteins; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D002843:Chromatin; D019925:Cyclin A; D056748:Cyclin H; D016213:Cyclins; D006657:Histones; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495891:Cdk2 protein, Xenopus; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase