The Human CDK8 Subcomplex Is a Histone Kinase That Requires Med12 for Activity and Can Function Independently of Mediator

Abstract

The four proteins CDK8, cyclin C, Med12, and Med13 can associate with Mediator and are presumed to form a stable “CDK8 subcomplex” in cells. We describe here the isolation and enzymatic activity of the 600-kDa CDK8 subcomplex purified directly from human cells and also via recombinant expression in insect cells. Biochemical analysis of the recombinant CDK8 subcomplex identifies predicted (TFIIH and RNA polymerase II C-terminal domain [Pol II CTD]) and novel (histone H3, Med13, and CDK8 itself) substrates for the CDK8 kinase. Notably, these novel substrates appear to be metazoan-specific. Such diverse targets imply strict regulation of CDK8 kinase activity. Along these lines, we observe that Mediator itself enables CDK8 kinase activity on chromatin, and we identify Med12—but not Med13—to be essential for activating the CDK8 kinase. Moreover, mass spectrometry analysis of the endogenous CDK8 subcomplex reveals several associated factors, including GCN1L1 and the TRiC chaperonin, that may help control its biological function. In support of this, electron microscopy analysis suggests TRiC sequesters the CDK8 subcomplex and kinase assays reveal the endogenous CDK8 subcomplex—unlike the recombinant submodule—is unable to phosphorylate the Pol II CTD.

ACKNOWLEDGMENTS

We thank Bob Roeder for providing the Med12 cDNA, Emma Lees for providing cDNAs for CDK8 and cyclin C, and Karolin Luger for cDNAs encoding core histones. The 5S G5E4 template was a gift from Jerry Workman.

This study was supported by the NCI P01 CA112181 (D.J.T.) and R01 CA117907 (J.M.E.) and in part by NIH grants T32 GM07135 and T32 GM065103 (M.T.K. and K.D.M.).