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J. Biol. Chem., Vol. 280, Issue 14, 14302-14309, April 8, 2005

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Molecular and Genetic Studies Imply Akt-mediated Signaling Promotes Protein Kinase CII Alternative Splicing via Phosphorylation of Serine/Arginine-rich Splicing Factor SRp40^*

Niketa A. Patel, Satoshi Kaneko, Hercules S. Apostolatos, Sun Sik Bae¶, James E. Watson||, Karen Davidowitz, David S. Chappell, Morris J. Birnbaum¶, Jin Q. Cheng, and Denise R. Cooper||**

From the Departments of Biochemistry and Molecular Biology and Internal Medicine and the Department of Pathology, University of South Florida College of Medicine, Tampa, Florida 33612, the ^||J. A. Haley Veterans Hospital, Tampa, Florida 33612, and the ^¶Howard Hughes Medical Institute, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Insulin regulates alternative splicing of PKCII mRNA by phosphorylation of SRp40 via a phosphatidylinositol 3-kinase pathway (Patel, N. A., Chalfant, C. E., Watson, J. E., Wyatt, J. R., Dean, N. M., Eichler, D. C., and Cooper, D. C. (2001) J. Biol. Chem. 276, 22648–22654). Transient transfection of constitutively active Akt2 kinase promotes PKCII exon inclusion. Serine/arginine-rich (SR) RNA-binding proteins regulating the selection of alternatively spliced exons are potential substrates of Akt kinase because many of them contain RXRXX(S/T) motifs. Here we show that Akt2 kinase phosphorylated SRp40 in vivo and in vitro. Mutation of Ser⁸⁶ on SRp40 blocked in vitro phosphorylation. In control Akt2(+/+) fibroblasts, insulin treatment increased the phosphorylation of endogenous SR proteins, but their phosphorylation state remained unaltered by insulin in fibroblasts from Akt2(-/-) mice. Levels of PKCII protein were up-regulated by insulin in Akt2(+/+) cells; however, only very low levels of PKCII were detected in Akt2(-/-) cells and did not change following insulin treatment. Endogenous PKCI and -II mRNA levels in Akt2(+/+) and Akt2(-/-) gastrocnemius muscle tissues were compared using quantitative real time PCR. The results indicated a 54% decrease in the expression of PKCII levels in Akt(-/-), whereas PKCI levels remained unchanged in both samples. Further, transfection of Akt2(-/-) cells with a PKCII splicing minigene revealed defective II exon inclusion. Co-transfection of the mutated SRp40 attenuated II exon inclusion. This study provides in vitro and in vivo evidence showing Akt2 kinase directly phosphorylated SRp40, thereby connecting the insulin, PI 3-kinase/Akt pathway with phosphorylation of a site on a nuclear splicing protein promoting exon inclusion. This model is upheld in Akt2-deficient mice with insulin resistance leading to diabetes mellitus.

Received for publication, October 8, 2004 , and in revised form, January 24, 2005.

^* This work was supported by NIDDK, National Institutes of Health Grant 54393 (to D. R. C.), the Department of Veterans Affairs Medical Research Service (to D. R. C.), National Institutes of Health Grant DK39615 (to M. J. B.), and National Institutes of Health Grant CA077935 (to J. Q. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Research Service 151, J. A. Haley Veterans Hospital, 13000 Bruce B. Downs Blvd., Tampa, FL 33612. Tel.: 813-972-2000, Ext. 7017; Fax: 813-978-5889; E-mail: dcooper{at}hsc.usf.edu.

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