Active site phosphorylation of CDK11 is antagonised by PNUTS‐PP1 and localised in the centrosomes
Authors: Abdulrahman Aljabri 1,2; A Campbell 1; D Byrne 1; P Eyers 1; C Eyers 1; D Bennett 1
Affiliations: 1) Institute of Systems, Molecular and Integrative Biology- University of Liverpool ; 2) College of Pharmacy- Taibah University
Keywords: h. kinase/phosphatase/cyclin; a. mitosis
Reversible protein phosphorylation is a post-translational modification (PTM) that plays a vital role in the regulation of cellular function and is controlled by protein kinases and phosphatases, which add and remove phosphate respectively. Serine/Threonine Protein Phosphatase 1 (PP1) has multiple and essential roles in cellular processes, including RNA splicing, cell division and metabolism of glycogen (Cohen, 2002). In vivo, PP1 catalytic activity is regulated by PP1 interacting proteins (PIPs), one of the most abundant of which is PP1 Nuclear Target Subunit (PNUTS). The PNUTS-PP1 holoenzyme has been implicated in many cellular functions including proliferation, DNA repair, and cell cycle progression and mRNA synthesis. To better understand the role of PNUTS/PP1 we recently conducted phosphoproteomics analysis to identify additional substrates of Drosophila PNUTS/PP1. We found that PNUTS/PP1 associates with and regulates the phosphorylation state of the protein kinase Pitslre/CDK11. CDK11 becomes hyper-phosphorylated in its active site at S712 (or S538 in a shorter isoform) when binding of PP1 to PNUTS is disrupted by a mutation in the PP1-binding site of PNUTS (W726A). S712 in CDK11 is equivalent to a serine residue in CDK7 that modulates CDK7 activity and binding to cyclin H (Larochelle et al., 2001; Martinez et al., 1997) (Endicott & Noble, 2013).
Our current focus is therefore to determine when and where CDK11 is phosphorylated. In Drosophila neuroblasts we found elevation of phosphorylated CDK11 (pCDK11) during mitosis, and enrichment of pCDK11 staining in centromeres. Overexpression of kinase dead forms of CDK11 resulted in short or aberrant mitotic spindles . To investigate in vivo function further, we generated non-phosphorylatable (S712/538A) and phosphomimic (S712/538E) CDK11 alleles by CRISPR-Cas9 mediated mutagenesis. We found that non-phosphorylatable CDK11 is recessive lethal in early larval stages, indicating that CDK11 phosphorylation is likely to play an essential role.
Here we will describe the results of our findings and discuss the possibility that pCDK11 modulates CDK11 activity during the cell cycle to promote mitotic spindle formation or function.