Analog-sensitive cell line identifies cellular substrates of CDK9

// Tim-Michael Decker 1 , 4 , Ignasi Forne 2 , Tobias Straub 3 , Hesham Elsaman 1 , Guoli Ma 1 , Nilay Shah 1 , 5 , Axel Imhof 2 and Dirk Eick 1 1 Department of Molecular Epigenetics, Helmholtz Center Munich and Center for Integrated Protein Science Munich (CIPSM), Germany 2 Biomedical Center Munich, ZFP, Ludwig-Maximilian University Munich, Germany 3 Bioinformatic Unit, Biomedical Center Munich, Ludwig-Maximilian University Munich, Planegg-Martinsried, Germany 4 Present address: Department of Biochemistry, University of Colorado, Boulder, USA 5 Present address: Stowers Institute for Medical Research, Kansas City, Missouri, USA Correspondence to: Tim-Michael Decker, email: timmichael.decker@colorado.edu Dirk Eick, email: eick@helmholtz-muenchen.de Keywords: CDK9; protein kinase; transcription; RNA splicing; phosphoproteomics Received: September 12, 2019     Accepted: November 07, 2019     Published: December 10, 2019 ABSTRACT Transcriptional cyclin-dependent kinases regulate all phases of transcription. Cyclin-dependent kinase 9 (CDK9) has been implicated in the regulation of promoter-proximal pausing of RNA polymerase II and more recently in transcription termination. Study of the substrates of CDK9 has mostly been limited to in vitro approaches that lack a quantitative assessment of CDK9 activity. Here we analyzed the cellular phosphoproteome upon inhibition of CDK9 by combining analog-sensitive kinase technology with quantitative phosphoproteomics in Raji B-cells. Our analysis revealed the activity of CDK9 on 1102 phosphosites quantitatively, and we identified 120 potential cellular substrates. Furthermore, a substantial number of CDK9 substrates were described as splicing factors, highlighting the role of CDK9 in transcription-coupled splicing events. Based on comparison to in vitro data, our findings suggest that cellular context fundamentally impacts the activity of CDK9 and specific selection of its substrates.