Splicing is a co-transcriptional process whereby a single gene can be converted into multiple unique mRNA fragments for enhanced protein diversity. While splicing is integral for normal cellular function in complex organisms, mistakes i splice-selection can be extremely deleterious. In fact, splicing errors are associated with numerous human diseases including muscular dystrophy, Alzheimer's disease, parkinsonism, metabolic disorders, ataxias and cancers. Splicing occurs at the spliceosome, a macromolecular complex that includes both RNA and proteins. In the latter group, SR proteins are essential splicing factors that control where and how the spliceosome assembles on precursor mRNA. SR proteins contain C-terminal domains rich in arginine-serine repeats whose polyphosphorylation controls splice-site selection. The SRPK family of protein kinases phosphorylates these RS domains directing SR proteins into the nucleus for splicing activity. While SRPKs are normally localized to the cytoplasm for this function, they can enter the nucleus under certain conditions further affecting SR protein phosphorylation levels and alternative gene splicing. Although phosphorylation is critical for splice-site choice, very little is known about how residue-specific phosphorylation of SR proteins controls alternative gene splicing. Clearly, knowing how the SRPKs are regulated in the cell and how they recognize and phosphorylate SR proteins is essential for an understanding of gene splicing and disease pathologies related to mis-splicing. We showed that SRPKs and phosphatases work in opposite directions concentrating phosphates toward the C-terminal end of RS domains. We will now explore how this unique phosphate distribution termed "phosphate biasing" affects SR protein function. We recently showed that the catalytic activity of SRPK1 is dependent on a nucleotide release factor composed of sequence elements from a large insert domain and an N-terminal extension. We propose that this conserved release factor is a hub for SRPK regulation in the cell. We will demonstrate how phosphorylation and protein-protein interactions modulate this factor and regulate SR protein phosphorylation levels and gene splicing.

Public Health Relevance

Incorrect splicing of genetic material is responsible for many neurodegenerative diseases and cancer. To better understand the connection between human disease and gene processing, we are studying the role of SRPKs, a critical enzyme family that regulates splicing activity in the cell.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM067969-10S1
Application #
8920239
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Bender, Michael T
Project Start
2004-02-01
Project End
2016-12-31
Budget Start
2014-01-01
Budget End
2014-12-31
Support Year
10
Fiscal Year
2014
Total Cost
$69,685
Indirect Cost
$24,727
Name
University of California San Diego
Department
Pharmacology
Type
Schools of Medicine
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Aubol, Brandon E; Adams, Joseph A (2014) Recruiting a silent partner for activation of the protein kinase SRPK1. Biochemistry 53:4625-34
Aubol, Brandon E; Jamros, Michael A; McGlone, Maria L et al. (2013) Splicing kinase SRPK1 conforms to the landscape of its SR protein substrate. Biochemistry 52:7595-605
Barkho, Sulyman; Pierce, Levi C T; McGlone, Maria L et al. (2013) Distal loop flexibility of a regulatory domain modulates dynamics and activity of C-terminal SRC kinase (csk). PLoS Comput Biol 9:e1003188
Aubol, Brandon E; Plocinik, Ryan M; Hagopian, Jonathan C et al. (2013) Partitioning RS domain phosphorylation in an SR protein through the CLK and SRPK protein kinases. J Mol Biol 425:2894-909
Sumanasekera, Chiranthani; Kelemen, Olga; Beullens, Monique et al. (2012) C6 pyridinium ceramide influences alternative pre-mRNA splicing by inhibiting protein phosphatase-1. Nucleic Acids Res 40:4025-39
Plocinik, Ryan M; Li, Sheng; Liu, Tong et al. (2011) Regulating SR protein phosphorylation through regions outside the kinase domain of SRPK1. J Mol Biol 410:131-45
Aubol, Brandon E; Adams, Joseph A (2011) Applying the brakes to multisite SR protein phosphorylation: substrate-induced effects on the splicing kinase SRPK1. Biochemistry 50:6888-900
Ghosh, Gourisankar; Adams, Joseph A (2011) Phosphorylation mechanism and structure of serine-arginine protein kinases. FEBS J 278:587-97
Ma, Chen-Ting; Ghosh, Gourisankar; Fu, Xiang-Dong et al. (2010) Mechanism of dephosphorylation of the SR protein ASF/SF2 by protein phosphatase 1. J Mol Biol 403:386-404
Jamros, Michael A; Oliveira, Leandro C; Whitford, Paul C et al. (2010) Proteins at work: a combined small angle X-RAY scattering and theoretical determination of the multiple structures involved on the protein kinase functional landscape. J Biol Chem 285:36121-8

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