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Identification and validation of CDK13 interacting proteins

University of British Columbia

Cyclin dependent kinases (CDKs) are components of signal transduction pathways that regulate cellular functions by phosphorylation of substrate proteins in response to upstream signals. The kinase domains of CDK12 and CDK13 are most similar to CDK9; CDK9 phosphorylates the C terminal domain (CTD) of RNA Polymerase II in order to stimulate processive transcription elongation. However, while most human CDKs consist of little more than a kinase domain, CDK12 and CDK13 are much larger and have several protein-protein interaction domains suggesting that they could participate within regulatory cascades. They also have a RS domain found in the SR protein family of splicing factors. Consistent with these features CDK12 and CDK13 co-localize with splicing factors and RNA Polymerase II in nuclear speckles. Based on these features CDK12 and CDK13 have been proposed to coordinately regulate splicing and transcription. Consistent with this hypothesis, both kinases phosphorylate the CTD of RNA polymerase II and regulate the alternative splicing of the Adenovirus E1a mini-gene model substrate. CDK12 has been found to interact with the splicing factors PRP19, CDC5L, RBM25, FBP11 and SRP55. Due to the similarity of CDK13 to CDK12, I investigated the interacting partners of CDK13 by immunoprecipitation and mass spectrometry and determined that CDK13 interacts with same splicing factors as CDK12. These interactions were validated by immunoprecipitation – western blot analysis. My results also indicated that PRP19 and CDC5L interact as a complex with CDK13. Therefore, the protein interaction partners of CDK13 and CDK12 suggest functional mechanisms for their ability to regulate splicing. In parallel projects, to begin investigating the functional roles of the kinase domain of CDK12 I constructed and expressed different CDK12 mutants in insect cells and in mammalian cells. Also to investigate the role of the CDK12 mutants and the protein-protein interactions of CDK13 in alternative splicing, I also developed a PCR based E1A mini-gene splicing assay.

Text

2012-08-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/43130

Medical Genetics

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/