The graphs are ratios of phospho-SR protein/SR protein (pSRp/SRp). promoting the pivotal role of both kinases in insulin-mediated RNA alternative splicing and glucose uptake. Alternative pre-mRNA splicing provides a versatile mechanism in gene expression and proteome diversity. It is also a means of diversifying signaling pathways when kinases, such as protein kinase C (PKC) , are the target genes. Phosphorylated serine/arginine-rich (SR) proteins function as exon BQU57 splicing enhancers and repressors within the spliceosomal complex. This family of proteins plays pivotal roles in constitutive and regulated splicing (1), because they are located BQU57 at the center of the regulatory pathways that lead to the selection of alternative splice sites. The various kinases that regulate the phosphorylation, intranuclear distribution, and protein-protein interactions of SR proteins play key roles in selection of alternative splice sites (1,2,3). Recently, Akt2 or PKB, an oncogene originally isolated from retrovirus AKT8-induced mouse thymic lymphoma, was shown to regulate alternative splicing (4,5). Additionally, SR protein-specific kinases (SRPK), CDC2-like kinases (Clk), cAMP-dependent protein kinase (PKA), and PKC all phosphorylate splicing factors containing Arg/Ser repeat motifs and have the potential to regulate splicing (6,7,8). The Clk kinases participate in a network of regulatory mechanisms that BQU57 enable SR proteins to control RNA splicing in response to phosphorylation (7,8,9,10). Clk/Sty kinase controls splicing during cellular proliferation and differentiation (11,12,13). Clk/Sty (Clk1) and Clk2, Clk3, and Clk4 are phosphorylated on serine/threonine and tyrosine residues (7), and they contain Akt-substrate motifs, implying that Clk BQU57 could be regulated by the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and that this alters their substrate specificity. However, no upstream kinase has been shown to regulate Clk/Sty function, thus limiting our understanding of its regulatory roles. The unique mechanism that underlies alternative splicing of PKC pre-mRNA has been well documented in mammalian cells and animal models (14,15,16,17); these studies implicated Clk/Sty in splicing of PKCII mRNA, a step necessary for insulin action BQU57 (18,19,20). Insulin stimulates the inclusion Spry2 of an exon encoding the C-terminal domain of PKCII; PKCI is the other splice variant. The pathway involves PI3K/Akt2-mediated phosphorylation of multiple SR proteins, including SRp40 (5). SR proteins, within their Arg/Ser domains, contain the highest number of Akt consensus motifs found in proteins (21). Regulation of alternative splicing and gene expression by PDK/Akt provides a unique modulation of growth factor action at the level of extracellular signaling (22,23,24). Because Clk/Sty contains two Akt consensus motifs, Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr), we hypothesized that its activity might be regulated by insulin and, furthermore, that Akt phosphorylation of Clk/Sty was necessary for the rules of PKCII exon inclusion. == Materials and Methods == == Materials == Tissue tradition medium was purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Norcross, GA). Porcine insulin was from Sigma Chemical Co. (St. Louis, MO). Reagents for polyacrylamide gel electrophoresis were from Bio-Rad (Hercules, CA). SRp75 polyclonal antibody was raised in rabbits to the synthetic peptide NH2-(GC)KDHAEDKLQNNDSAGKAK-COOH (residues 119136), SRp30b/SC35 antibody to the synthetic peptide NH2-CRRVGDVYIPRDRYTKE-COOH (residues 3853), and SRp55 antibody to the synthetic peptide NH2-CGERVIVEHARGPRRDRD-COOH (residues 6178) by Bio-Synthesis, Inc. (Louisville, TX). Antibodies were characterized alongside unreactive preimmune antisera and were shown to recognize rat, mouse, and human being cell line proteins. Antibody to a phospho-epitope in all SR proteins (mAb104) was from hybridoma cells (CRL 2067; American Type Tradition Collection, Manassas, VA). Anti-phospho-Akt (Ser473 and Akt antibody) and anti-Akt substrate (RXRXXS/T) antibody were from Cell Signaling (Beverly, MA). Anti-Clk/Sty antibody was kindly provided by Dr. Wayne Manley (Columbia University or college, New York, NY). Enhanced chemiluminescence reagents were from Pierce (Rockford, IL).TaqPlatinum polymerase was from PerkinElmer Existence Sciences (Norwalk, CT). Lipofectin was from Invitrogen. PI3K inhibitor LY294002 and Src kinase inhibitor PP1 were purchased from Calbiochem (La Jolla, CA). All other chemicals and reagents were purchased from the usual vendors. PP1 is definitely preincubated with cells.