T-loop phosphorylation stabilizes the CDK7-cyclin H-MAT1 complex in vivo and regulates its CTD kinase activity.
Cyclin-dependent kinase (CDK)7-cyclin H, the CDK-activating kinase (CAK) and TFIIH-associated kinase in metazoans can be activated in vitro through T-loop phosphorylation or binding to the RING finger protein MAT1. Although the two mechanisms can operate independently, we show that in a physiological setting, MAT1 binding and T-loop phosphorylation cooperate to stabilize the CAK complex of Drosophila. CDK7 forms a stable complex with cyclin H and MAT1 in vivo only when phosphorylated on either one of two residues (Ser164 or Thr170) in its T-loop. Mutation of both phosphorylation sites causes temperature-dependent dissociation of CDK7 complexes and lethality. Furthermore, phosphorylation of Thr170 greatly stimulates the activity of the CDK7- cyclin H-MAT1 complex towards the C-terminal domain of RNA polymerase II without significantly affecting activity towards CDK2. Remarkably, the substrate-specific increase in activity caused by T-loop phosphorylation is due entirely to accelerated enzyme turnover. Thus phosphorylation on Thr170 could provide a mechanism to augment CTD phosphorylation by TFIIH-associated CDK7, and thereby regulate transcription.
Larochelle
S
S
Cell Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA.
Chen
J
J
Knights
R
R
Pandur
J
J
Morcillo
P
P
Erdjument-Bromage
H
H
Tempst
P
P
Suter
B
B
Fisher
R P
RP
D000595:Amino Acid Sequence; D000818:Animals; D001704:Biopolymers; D056748:Cyclin H; D018844:Cyclin-Dependent Kinases; D016213:Cyclins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004330:Drosophila; D029721:Drosophila Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013379:Substrate Specificity; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D001704:Biopolymers; C534754:CycH protein, Drosophila; D056748:Cyclin H; D016213:Cyclins; D029721:Drosophila Proteins; D011494:Protein Kinases; C060137:carboxy-terminal domain kinase; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase