UC San Diego

The SR protein family of splicing factors plays critical roles in many facets of RNA metabolism such as constitutive and alternative splicing, mRNA export, and transcription. SR proteins are carefully regulated by phosphorylation in vivo. Phosphorylation and dephosphorylation events influence protein-protein interactions that determine SR protein localization and recruitment to and from active sites of transcription and translation in the cell. Phosphorylation of SR proteins is an important regulatory mechanism of gene expression at the post-transcriptional level. The SR proteins and the kinases that regulate them are linked to multiple neurodegenerative disease states and cancer. Therefore, detailed understanding of SR kinase catalysis with SR proteins is necessary to elucidate biological mechanisms of regulation and to provide a foundation for novel therapeutic modalities. The finding that spacer deleted SRPK1 processively phosphorylates its substrate ASF/SF2 has important implications for the biological activity of this splicing factor in mammalian cells. We have reproduced this result with full-length independently purified components in a novel efficient start-trap system that utilizes a catalytically inactive form of the kinase. We show that like SRPK1, phosphorylation of ASF/SF2 by Clk /Sty occurs in a processive manner. The SRPK1 spacer domain was found to have minimal contribution to kinetic and thermodynamic properties with its substrate. We determined that SRPK1 preferentially phosphorylates the N- terminal portion of the ASF/SF2 RS domain, and that SRPK1 and Clk/Sty cooperate to completely phosphorylate this region of the splicing factor.To investigate the unusual high affinity of SRPK1 for its substrate, we analyzed multiple domains of ASF/SF2 by developing a competitive inhibition assay from which equilibrium dissociation constants could be determined in the form KI. We discovered that the kinase makes low nano-molar contacts with residues in both the RRM and RS domains of ASF/SF2. The absence of additive thermodynamics in the context of SRPK1 interaction with full-length ASF/SF2 indicates that the kinase utilizes sub-optimal recognition of its substrate during catalysis to allow the flexibility required for poly-phosphorylation of the RS domain. The final studies of this thesis employed high-resolution FT- ICRMS to refine our understanding of ASF/SF2 phosphorylation by SRPK1. We report that SRPK1 phosphorylates 14-15 residues of ASF/SF2, and that this endpoint did not change for RRM deletion constructs. We demonstrate mechanistic changes that reflect enzyme kinetics with an aggregated substrate. And finally, we show that the phospho-serine observed in the SRPK1[Delta]NS:ASF/SF2[Delta]RRM1 crystal structure was due to ATP contaminated AMPPNP. A result that significantly furthered insights gained from this complex structure