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J Mol Biol. 2013 Aug 23;425(16):2894-909. doi: 10.1016/j.jmb.2013.05.013. Epub 2013 May 23.

Partitioning RS domain phosphorylation in an SR protein through the CLK and SRPK protein kinases.

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Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093-0636, USA.


SR proteins are essential splicing factors whose biological function is regulated through phosphorylation of their C-terminal RS domains. Prior studies have shown that cytoplasmic-nuclear translocalization of the SR protein SRSF1 is regulated by multisite phosphorylation of a long Arg-Ser repeat in the N-terminus of the RS domain while subnuclear localization is controlled by phosphorylation of a shorter Arg-Ser repeat along with several Ser-Pro dipeptides in the C-terminus of the RS domain. To better understand how these two kinases partition Arg-Ser versus Ser-Pro specificities, we monitored the phosphorylation of SRSF1 by CLK1 and SRPK1. Although SRPK1 initially binds at the center of the RS domain phosphorylating in an orderly, N-terminal direction, CLK1 makes widespread contacts in the RS domain and generates multiple enzyme-substrate complexes that induce a random addition mechanism. While SRPK1 rapidly phosphorylates N-terminal serines, SRPK1 and CLK1 display similar activities toward Arg-Ser repeats in the C-terminus, suggesting that these kinases may not separate function in a strict linear manner along the RS domain. CLK1 induces a unique gel shift in SRSF1 that is not the result of enhanced Arg-Ser phosphorylation but rather is the direct result of the phosphorylation of several Ser-Pro dipeptides. These prolines are important for binding and phosphorylation of the SR protein by CLK1 but not for the SRPK1-dependent reaction. The data establish a new view of SR protein regulation in which SRPK1 and CLK1 partition activities based on Ser-Pro versus Arg-Ser placement rather than on N- and C-terminal preferences along the RS domain.


BSA; CLK; Cdc2-like kinase; NIH; National Institutes of Health; RNA recognition motif; RRM; SR protein; SR-specific protein kinase; SRPK; bovine serum albumin; kinase; kinetics; phosphorylation; splicing

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