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

Abstract

Insulin regulates splicing of pre-mRNA for protein kinase C␤II (PKC␤II) 1 by enhanced exon inclusion in rat skeletal muscle myotubes (L6) (1, 2). Alternative splicing of nuclear pre-mRNA allows for the formation of multiple RNA isoforms from a single primary RNA transcript. It is a widespread mechanism controlling gene expression in higher eukaryotes (3), and an estimated 30,000 human genes generate over 100,000 protein isoforms with distinct functions by alternative splicing (4, 5). Alternative splicing can be controlled developmentally or by tissue specificity (6 -9) and is a well studied mechanism. Our laboratory has demonstrated hormonal regulation of PKC␤II alternative splicing, and mechanisms for exon recognition involve the regulated phosphorylation of the serine/arginine-rich (SR) protein SRp40 (10, 11).A family of splicing factors, SR proteins, are highly phosphorylated RNA-binding proteins involved in the alternative splicing process (12-14). Ten highly conserved SR proteins consist of one or two N-terminal ribonucleoprotein-type RNA-binding domains and a C-terminal SR domain that promotes proteinprotein interactions within the splicing complex. SR proteins are required for constitutive splicing for the formation of the early prespliceosomal complex to stabilize U1 small nuclear ribonucleoprotein at the 5Ј splice site and in spliceosome formation to mediate interactions between U1 small nuclear ribonucleoprotein and U2AF, bound to the 5Ј and 3Ј splice sites (15)(16)(17). SR proteins also function in alternative splicing in vivo in a concentration-dependent manner by modulating 5Ј splice site choice (18,19). The SR domains direct SR proteins to localize to speckles within the nucleus (20), and some SR proteins can "shuttle" between the nucleus and cytoplasm depending on their phosphorylation state (21). The function of SR proteins in hormone-regulated alternative splicing has not been elucidated.Our studies focused on SRp40 because its expression was known to be regulated by insulin (22). Predictions by SELEX and reports by other groups identified a binding site for SRp40 in the downstream intron of the rat PKC␤ gene (23). We found that modified antisense oligonucleotides (RNase H-resistant) blocked the interaction of SRp40 with the PKC␤II intron and

Subtle distinct regulations of late erythroid molecular events by PI3K/AKT-mediated activation of Spi-1/PU.1 oncogene autoregulation loop

“…Recent works have provided in vitro and in vivo evidence showing that PI3K/AKT pathway regulates specific alternative splicing events through phosphorylation of selective SR proteins (Blaustein et al, 2005;Patel et al, 2005). Here, we provide the first observation that couples a functionally critical alternative splicing event to a signaling cascade that has an essential role in Epo-induced erythropoiesis.…”

Serine Arginine Splicing Factor 3 Is Involved in Enhanced Splicing of Glucose-6-phosphate Dehydrogenase RNA in Response to Nutrients and Hormones in Liver

“…In mammalian cells, insulin regulates alternative splicing of PKC␤II (45,46). The effect of insulin upon alternative splicing of PKC␤II is coincident with an increase in the phosphorylation of SRSF5 (11). In hepatocytes in culture, insulin increases G6PD mRNA and increases SR protein phosphorylation, including phosphorylation of SRSF3 (Fig.…”

Dual-specificity Tyrosine Phosphorylation-regulated Kinase 1A (Dyrk1A) Modulates Serine/Arginine-rich Protein 55 (SRp55)-promoted Tau Exon 10 Inclusion

“…To date, multiple kinases, including SR protein kinase 1 (SRPK1) (36), SRPK2 (37), Cdc-like kinase (Clk/Sty) (38), DNA topoisomerase I (39), cAMP-dependent protein kinase (PKA), and AKT (40,41), have been shown to phosphorylate the RS domain of SF2/ASF. We recently found that Dyrk1A phosphorylates SF2/ASF, SC35, and 9G8 and regulates both subcellular localization and activity (13,14,19).…”