Manipulation of alternative splicing by a newly developed inhibitor of Clks.

Jun Koizumi,Takamitsu Hosoya,Kiyoshi Furuichi,Michael V Murray,Michiko Muraki,Hiroshi Shibuya,Kengo Sumi,Bisei Ohkawara,Hiroshi Kimura,Hiroshi Onogi,Tomonobu Koizumi,Masatoshi Hagiwara,Jun-Ichiro Yomoda,Masaaki Suzuki,Adrian R Krainer

doi:10.1074/jbc.m314298200

Abstract

The regulation of splice site usage provides a versatile mechanism for controlling gene expression and for the generation of proteome diversity, playing an essential role in many biological processes. The importance of alternative splicing is further illustrated by the increasing number of human diseases that have been attributed to mis-splicing events. Appropriate spatial and temporal generation of splicing variants demands that alternative splicing be subjected to extensive regulation, similar to transcriptional control. The Clk (Cdc2-like kinase) family has been implicated in splicing control and consists of at least four members. Through extensive screening of a chemical library, we found that a benzothiazole compound, TG003, had a potent inhibitory effect on the activity of Clk1/Sty. TG003 inhibited SF2/ASF-dependent splicing of beta-globin pre-mRNA in vitro by suppression of Clk-mediated phosphorylation. This drug also suppressed serine/arginine-rich protein phosphorylation, dissociation of nuclear speckles, and Clk1/Sty-dependent alternative splicing in mammalian cells. Consistently, administration of TG003 rescued the embryonic defects induced by excessive Clk activity in Xenopus. Thus, TG003, a novel inhibitor of Clk family will be a valuable tool to dissect the regulatory mechanisms involving serine/arginine-rich protein phosphorylation signaling pathways in vivo, and may be applicable for the therapeutic manipulation of abnormal splicing.

Highlights

The regulation of splice site usage provides a versatile mechanism for controlling gene expression and for the generation of proteome diversity, playing an essential role in many biological processes
The overexpression of Clks affects splicing site selection of pre-mRNA of both its own transcript and adenovirus E1A transcripts in vivo [17]. These results have led us to the current model that Clk family members regulate alternative splicing by phosphorylation of SR proteins, their signal pathways and biological functions are largely unknown in vertebrates
TG003 Inhibits Clk1/Sty and Clk4 in Vitro—Through extensive screening of 100,000 chemical compounds in a chemical library by in vitro phosphorylation assay, we found that a benzothiazole compound had a potent inhibitory effect on the activity of Clk1/Sty

Summary

EXPERIMENTAL PROCEDURES

Synthesis of TG003—A series of benzothiazole compounds including TG003 were synthesized according to the procedures reported by Gupta et al [27]. In Vitro Kinase Assay—Kinase activity of Clks and SRPKs was assayed in a reaction mixture, containing 200 mM Tris-HCl (pH 7.5), 12.5 mM MgCl2, 8 mM dithiothreitol, 4 mM EGTA, 1–20 ␮M ATP, 1 ␮Ci of [␥-32P]ATP, 1 ␮g of synthetic peptide of SF2/ASF RS domain (NH2RSPSYGRSRSRSRSRSRSRSRSNSRSRSY-OH), and 0.1–1 ␮g of purified kinases in a final volume of 40 ␮l. CAMP-dependent protein kinase activity was assayed in a reaction mixture containing 80 mM Tris-HCl (pH 7.5), 12.5 mM MgCl2, 8 mM dithiothreitol, 4 mM EGTA, 10 ␮M ATP, 1 ␮Ci of [␥-32P]ATP, 5 ␮g of histone H1 (Sigma), and 1 ␮g of catalytic subunit of rat cAMP-dependent protein kinase purified as described [30].

Synthetic Inhibitor of Clks and Splicing

RESULTS

DISCUSSION