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Investigation of substrate phosphorylation by and subcellular localization of SR protein kinase 1


In eukaryotic organisms, mRNA synthesis requires splicing to remove non-coding introns. Two types of splicing events are constitutive and alternative. The serine-arginine (SR) protein and SR-protein kinase (SRPK) families are the two protein families necessary for alternative splicing. SR- proteins are characterized by N-terminal RNA recognition motif and C-terminal RS domain. In the cytoplasm, SRPK1, one of the SRPK family kinases, processively phosphorylates the RS in ASF/SF2, a well-studied SR- protein. The various phosphorylated forms of SR-proteins play critical role in alternative splicing. The focus of my thesis work is how SRPK1 processively phosphorylates ASF/SF2 and what structural elements in SRPK1 regulate its subcellular localization. X-ray structures of the SRPK1: ASF/SF2 complex allowed us to propose two possible docking motifs in ASF/SF2: the C-terminal end of RRM and the N'- terminal RS. Using chemical cross-linking experiments, I have demonstrated that ASF/SF2 indeed possesses two docking motifs that mediate in sequential and phosphorylation-dependent manner. These experiments further established the C to N-terminal directionality of ASF/SF2 phosphorylation. I have further investigated the mechanism of how SRPK1 is distributed in cells. My experiments supported the existence of SRPK1 as a dimer and that the dimerization of SRPK1 needs its spacer domain as well as the C-terminal end. However, I found dimerization not to be the only factor in the regulation of cellular distribution of SRPK1. I have further shown that another splicing-related kinase family, Clk/Sty, which is exclusively nuclear, interacts with SRPK1 in vivo. It is likely that interactions with Clk/Sty may regulate the compartmentalization of SRPK1

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