Dual phosphorylation of the T-loop in cdk7: its role in controlling cyclin H binding and CAK activity.
A cyclin-dependent kinase (cdk)-activating kinase (CAK) has been shown previously to catalyze T-loop phosphorylation of cdks in most eukaryotic cells. This enzyme exists in either of two forms: the major one contains cdk7, cyclin H and an assembly factor called MAT-1, whilst the minor one lacks MAT-1. Cdk7 is unusual among cdks because it contains not one but two residues (S170 and T176 in Xenopus cdk7) in its T-loop that are phosphorylated in vivo. We have investigated the role of S170 and T176 phosphorylation in the assembly and activity of cyclin H-cdk7 dimers. In the absence of MAT-1, phosphorylation of the T-loop appears to be required for cdk7 to bind cyclin H. Phosphorylation of both residues does not require cyclin H binding in vitro. Phosphorylation of S170 is sufficient for cdk7 to bind cyclin H with low affinity, but high affinity binding requires T176 phosphorylation. By mutational analysis, we demonstrate that in addition to its role in promotion of cyclin H binding, S170 phosphorylation plays a direct role in the control of CAK activity. Finally, we show that dual phosphorylation of S170 and T176, or substitution of both phosphorylatable residues by aspartic residues, is sufficient to generate CAK activity to one-third of its maximal value in vitro, even in the absence of cyclin H and MAT-1, and may thus provide further clues as to how cyclins activate cdk subunits.
Martinez
A M
AM
Centre de Recherches de Biochimie Macromoléculaire, CNRS ERS155 and INSERM U 249, Montpellier, France.
Afshar
M
M
Martin
F
F
Cavadore
J C
JC
Labbé
J C
JC
Dorée
M
M
D000818:Animals; D042846:CDC2-CDC28 Kinases; D002850:Chromatography, Gel; D056748:Cyclin H; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D004789:Enzyme Activation; D008958:Models, Molecular; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000737:chemistry*; D010766:Phosphorylation; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011817:Rabbits; D012694:Serine; D013329:Structure-Activity Relationship; D013912:Threonine; D014981:Xenopus; D029867:Xenopus Proteins; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D056748:Cyclin H; D016213:Cyclins; D029867:Xenopus Proteins; D013912:Threonine; D012694:Serine; 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