Interaction of Cyclin-Dependent Kinase 12/CrkRS With Cyclin K1 Is Required for the Phosphorylation of the C-terminal Domain of RNA Polymerase II

Mol Cell Biol. 2012 Nov;32(22):4691-704. doi: 10.1128/MCB.06267-11. Epub 2012 Sep 17.

Abstract

CrkRS (Cdc2-related kinase, Arg/Ser), or cyclin-dependent kinase 12 (CKD12), is a serine/threonine kinase believed to coordinate transcription and RNA splicing. While CDK12/CrkRS complexes were known to phosphorylate the C-terminal domain (CTD) of RNA polymerase II (RNA Pol II), the cyclin regulating this activity was not known. Using immunoprecipitation and mass spectrometry, we identified a 65-kDa isoform of cyclin K (cyclin K1) in endogenous CDK12/CrkRS protein complexes. We show that cyclin K1 complexes isolated from mammalian cells contain CDK12/CrkRS but do not contain CDK9, a presumed partner of cyclin K. Analysis of extensive RNA-Seq data shows that the 65-kDa cyclin K1 isoform is the predominantly expressed form across numerous tissue types. We also demonstrate that CDK12/CrkRS is dependent on cyclin K1 for its kinase activity and that small interfering RNA (siRNA) knockdown of CDK12/CrkRS or cyclin K1 has similar effects on the expression of a luciferase reporter gene. Our data suggest that cyclin K1 is the primary cyclin partner for CDK12/CrkRS and that cyclin K1 is required to activate CDK12/CrkRS to phosphorylate the CTD of RNA Pol II. These properties are consistent with a role of CDK12/CrkRS in regulating gene expression through phosphorylation of RNA Pol II.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Cyclin-Dependent Kinases / antagonists & inhibitors
  • Cyclin-Dependent Kinases / genetics
  • Cyclin-Dependent Kinases / metabolism*
  • Cyclins / antagonists & inhibitors
  • Cyclins / genetics
  • Cyclins / metabolism*
  • Gene Expression Regulation*
  • Genes, Reporter
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Immunoprecipitation
  • Luciferases
  • Mass Spectrometry
  • Phosphorylation
  • Protein Binding
  • Protein Isoforms / antagonists & inhibitors
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism
  • Protein Structure, Tertiary
  • RNA Polymerase II / genetics
  • RNA Polymerase II / metabolism*
  • RNA, Small Interfering / genetics
  • Signal Transduction / genetics

Substances

  • CCNK protein, human
  • Cyclins
  • Protein Isoforms
  • RNA, Small Interfering
  • Luciferases
  • CDK12 protein, human
  • Cyclin-Dependent Kinases
  • RNA Polymerase II