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) an d TFIIH-associated kinase in metazoans can be activated in vitro through T- loop phosphorylation or binding to the RING finger protein MAT1. Although t he two mechanisms can operate independently, we show that in a physiologica l setting, MAT1 binding and T-loop phosphorylation cooperate to stabilize t he 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 (Ser16 4 or Thr170) in its T-loop. Mutation of both phosphorylation sites causes t emperature-dependent dissociation of CDK7 complexes and lethality. Furtherm ore, phosphorylation of Thr170 greatly stimulates the activity of the CDK7- cyclin H-MAT1 complex towards the C-terminal domain of RNA polymerase II wi thout significantly affecting activity towards CDK2. Remarkably, the substr ate-specific increase in activity caused by T-loop phosphorylation is due e ntirely to accelerated enzyme turnover. Thus phosphorylation on Thr170 coul d provide a mechanism to augment CTD phosphorylation by TFIIH-associated CD K7, and thereby regulate transcription.