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.
Highlights
Phosphorylation by protein kinases is a major post-translational modification in cell signaling
Our list of Cyclin-dependent kinase 9 (CDK9) substrates did not contain several of those substrates, that are mostly linked with the canonical role of CDK9, including Pol RNA polymerase II (II) C-terminal repeat domain (CTD), NELF, and DSIF
IWS1 was identified as a CDK9 substrate in vitro [11], validating this factor as a high confidence substrate of CDK9
Summary
Phosphorylation by protein kinases is a major post-translational modification in cell signaling. Higher eukaryotes encode for 518 putative protein kinases and many of them are expressed in cells at the same time [1]. While there are hundreds of kinases, only three amino acids, serine, threonine, and tyrosine, undergo modification by kinases in eukaryotes [2]. Analysis of the sequence and structure of phosphorylation sites has revealed only limited specificity in vivo and in vitro [3]. Given the large number of kinases and their limited specificity, protein phosphorylation apparently undergoes several layers of regulation. Recruitment of kinases and control of their activity substantially contribute to the regulation of protein phosphorylation in vivo [4]
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