Abstract
We asked whether the C-terminal repeat domain (CTD) kinase, CDK12/CyclinK, phosphorylates substrates in addition to the CTD of RPB1, using our CDK12analog-sensitive HeLa cell line to investigate CDK12 activity-dependent phosphorylation events in human cells. Characterizing the phospho-proteome before and after selective inhibition of CDK12 activity by the analog 1-NM-PP1, we identified 5,644 distinct phospho-peptides, among which were 50 whose average relative amount decreased more than 2-fold after 30 min of inhibition (none of these derived from RPB1). Half of the phospho-peptides actually showed >3-fold decreases, and a dozen showed decreases of 5-fold or more. As might be expected, the 40 proteins that gave rise to the 50 affected phospho-peptides mostly function in processes that have been linked to CDK12, such as transcription and RNA processing. However, the results also suggest roles for CDK12 in other events, notably mRNA nuclear export, cell differentiation and mitosis. While a number of the more-affected sites resemble the CTD in amino acid sequence and are likely direct CDK12 substrates, other highly-affected sites are not CTD-like, and their decreased phosphorylation may be a secondary (downstream) effect of CDK12 inhibition.
Highlights
Human CDK12, a tumor suppressor for ovarian and other cancers [1,2], is the catalytic subunit of the protein kinase complex CDK12/CyclinK [3,4]
We previously showed that inhibiting CDK12 activity in living cells leads rapidly to altered phosphorylation states of the RNAPII C-terminal repeat domain (CTD), and we demonstrate that CDK12 inhibition alters phosphorylation states of several dozen other proteins
From inspecting the amino acid sequences of the affected phosphorylation sites, we conclude that some are phosphorylated directly by CDK12, while others are phosphorylated by different kinases and are affected indirectly by CDK12 inhibition; the normal phosphorylation levels at all of the sites depend on CDK12 catalytic activity
Summary
Human CDK12, a tumor suppressor for ovarian and other cancers [1,2], is the catalytic subunit of the protein kinase complex CDK12/CyclinK [3,4]. Mammalian CDK12 has been connected with expression of genes involved in DNA damage responses [4,19], it has been implicated in mRNA 30 end formation [20,21], and CDK12 and its paralog CDK13 have been implicated in RNA processing and in expression of RNA processing factors [19]. Related to these latter observations, CDK12 and CDK13 have been shown biochemically to bind a number of RNA processing factors [3,19]. CDK12 and CDK13 have been shown to be important for mouse Embryonic Stem Cells (ESC) self-renewal [22] and for Biomolecules 2019, 9, 634; doi:10.3390/biom9100634 www.mdpi.com/journal/biomolecules
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