Interrogating the role and regulation of CDK11 active site phosphorylation

Abstract

Reversible protein phosphorylation is a post-translational modification (PTM) that is vital in regulating cellular function and is controlled by protein kinases and phosphatases. Protein Phosphatase 1 (PP1) is one of the major Serine/Threonine protein phosphatases necessary for RNA splicing, cell division, and glycogen metabolism (Cohen, 2002). In vivo, PP1 catalytic activity is regulated by PP1 interacting proteins (PIPs), including the PP1 Nuclear Target Subunit (PNUTS). The Drosophila PNUTS/PP1 holoenzyme has recently been identified to regulate the protein kinase pitslre/CDK11 (Campbell, 2018). Specifically, when PP1-binding to PNUTS was disrupted by a canonical PP1-binding site mutation, Pitslre/CDK11 became hyper-phosphorylated at S712 (or S538 in the short isoform) in PNUTS precipitates. This result suggests that Pitslre/CDK11 may be an in vivo substrate of PNUTS/PP1. I aimed in this study first to determine the effect of CDK11 phosphorylation (S538/712) on CDK11 activity. This also prompted us to determine when and where CDK11 is normally phosphorylated. Lastly, I aimed to investigate whether CDK11 S538/712 phosphorylation affects CDK11 activity using mutational analysis of the endogenous CDK11 allele.