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Insulin Regulates Alternative Splicing of Protein Kinase C βII through a Phosphatidylinositol 3-Kinase-dependent Pathway Involving the Nuclear Serine/Arginine-rich Splicing Factor, SRp40, in Skeletal Muscle Cells*

Open AccessPublished:June 22, 2001DOI:https://doi.org/10.1074/jbc.M101260200
      Insulin regulates the inclusion of the exon encoding protein kinase C (PKC) βII mRNA. In this report, we show that insulin regulates this exon inclusion (alternative splicing) via the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway through the phosphorylation state of SRp40, a factor required for insulin-regulated splice site selection for PKCβII mRNA. By taking advantage of a well known inhibitor of PI 3–kinase, LY294002, we demonstrated that pretreatment of L6 myotubes with LY294002 blocked insulin-induced PKCβII exon inclusion as well as phosphorylation of SRp40. In the absence of LY294002, overexpression of SRp40 in L6 cells mimicked insulin-induced exon inclusion. When antisense oligonucleotides targeted to a putative SRp40-binding sequence in the βII-βI intron were transfected into L6 cells, insulin effects on splicing and glucose uptake were blocked. Taken together, these results demonstrate a role for SRp40 in insulin-mediated alternative splicing independent of changes in SRp40 concentration but dependent on serine phosphorylation of SRp40 via a PI 3-kinase signaling pathway. This switch in PKC isozyme expression is important for increases in the glucose transport effect of insulin. Significantly, insulin regulation of PKCβII exon inclusion occurred in the absence of cell growth and differentiation demonstrating that insulin-induced alternative splicing of PKCβII mRNA in L6 cells occurs in response to a metabolic change.
      PKC
      protein kinase C
      PI 3-kinase
      phosphatidylinositol-3 kinase
      SR
      serine-arginine-rich
      RS
      arginine/serine domain
      HRS
      hepatic Arg-Ser protein
      RT-PCR
      reverse transcriptase-polymerase chain reaction
      AS
      antisense oligonucleotides
      BSA
      bovine serum albumin
      PAGE
      polyacrylamide gel electrophoresis
      α-MEM
      α-minimum Eagle's medium
      bp
      base pairs
      PCR
      polymerase chain reaction
      Insulin regulates levels of protein kinase C (PKC)1 βII mRNA in skeletal muscle by a novel mechanism that signals the activation of splice sites in the last intron of the pre-mRNA. Exon inclusion results in an mRNA that now encodes the C-terminal region of PKCβII affecting subcellular localization and substrate specificity of the kinase. The terminal PKCβI-specific exon with its 3′-untranslated region is spliced to the PKCβII-specific exon via exon inclusion such that a stop codon is introduced at the splice site, and as a result, the PKCβI exon becomes part of an extended 3′-untranslated region of PKCβII mRNA (
      • Ono Y.
      • Kikkawa U.
      • Ogita K.
      • Fujii T.
      • Kurokawa T.
      • Asaoka Y.
      • Sekiguchi K.
      • Ase K.
      • Igarashi K.
      • Nishizuka Y.
      ,
      • Ono Y.
      • Kurokawa T.
      • Fujii T.
      • Kawahara K.
      • Igarashi K.
      • Kikkawa U.
      • Ogita K.
      • Nishizuka Y.
      ). Therefore, PKCβII and PKCβI differ only by their C-terminal 52–50 amino acids, respectively. In contrast to PKCβI, increased expression of PKCβII results in activation/inactivation of the mitogen-activated kinase cascade (
      • Wang J.M.
      • Chao J.R.
      • Chen W.
      • Kuo M.L.
      • Yen J.J.
      • Yang-Yen H.F.
      ), glycogen kinase synthase 3β (
      • Goode N.
      • Hughes K.
      • Woodgett J.R.
      • Parker P.J.
      ), TLS/Fus (
      • Perrotti D.
      • Bonatti S.
      • Trotta R.
      • Martinez R.
      • Skorski T.
      • Salomoni P.
      • Grassilli E.
      • Iozzo R.V.
      • Cooper D.R.
      • Calabretta B.
      ), insulin receptor signaling (
      • Bossenmaier B.
      • Mosthaf L.
      • Mischak H.
      • Ullrich A.
      • Haring H.U.
      ), cyclin-dependent kinase (CDK)-activating kinase,
      Acevedo-Duncan, M., Patel, R., and Whelan, S., (2001) Cell Prolif., in press.
      2Acevedo-Duncan, M., Patel, R., and Whelan, S., (2001) Cell Prolif., in press.
      as well as cell proliferation (
      • Yamamoto M.
      • Acevedo-Duncan M.
      • Chalfant C.E.
      • Patel N.A.
      • Watson J.E.
      • Cooper D.R.
      ,
      • Yamamoto M.
      • Patel N.A.
      • Taggart J.
      • Sridhar R.
      • Cooper D.R.
      ,
      • Gui J.-F.
      • Lane W.S.
      • Fu X.-D.
      ), protein trafficking (
      • Disatnik M.-H.
      • Buraggi G.
      • Mochly-Rosen D.
      ), apoptosis, and glucose transport (
      • Henry R.R.
      • Ciaraldi T.P.
      • Mudaliar S.
      • Abrams L.
      • Nikoulina S.E.
      ,
      • Mistell T.
      • Caceres J.F.
      • Spector D.L.
      ).
      Pre-mRNA splicing occurs on nuclear spliceosomes, a macromolecular complex consisting of small nuclear ribonucleoproteins, proteins associated with heterogeneous nuclear RNA, and other splicing factors including serine-arginine-rich (SR) proteins (
      • Kramer A.
      ,
      • Will C.L.
      • Luhrmann R.
      ). Exon splicing is highly regulated, and numerous consensus sequences that bind specific factors participate in the control of tissue-specific or developmentally controlled splicing via SR protein-RNA and protein-protein interactions (
      • Lopez A.J.
      ). SR and SR-like proteins are characterized by a modular composition with one or more RNA recognition motifs and an arginine and serine domain (RS domain) in which the serine residues can be highly phosphorylated. The RS domain is responsible for protein-protein interactions and nuclear localization (
      • Fu X.D.
      ,
      • Valcarcel J.
      • Gaur R.K.
      • Singh R.
      • Green M.R.
      ,
      • Wu J.Y.
      • Maniatis T.
      ). SR and SR-like proteins have been implicated in 5′-splice site recognition and in the communication of splice sites caused by a network of SR proteins (
      • Yeakley J.M.
      • Morfin J.-P.
      • Rosenfeld M.G.
      • Fu X.-D.
      ). They can bind to exon enhancer motifs that are often purine-rich sequences that promote the use of suboptimal splice sites (
      • Fu X.-D.
      ). Their interaction with exon enhancers results in a concentration-dependent influence on alternative splicing (
      • Caceres J.F.
      • Stamm S.
      • Helfman D.M.
      • Krainer A.R.
      ,
      • Wang J.
      • Takagaki Y.
      • Manley J.L.
      ,
      • Screaton G.R.
      • Caceres J.F.
      • Mayeda A.
      • Bell M.V.
      • Plebanski M.
      • Jackson D.G.
      • Bell J.I.
      • Krainer A.R.
      ,
      • Diamond R.H.
      • Du K.
      • Lee V.M.
      • Mohn K.L.
      • Haber B.A.
      • Tewar D.S.
      • Taub R.
      ). Several SR protein kinases have been reported, including a U1 snRNP 70K-associated kinase, SR protein kinase (SRPK1), lamin B receptor kinase, and a family of CDC2-like kinases (
      • Gui J.-F.
      • Lane W.S.
      • Fu X.-D.
      ,
      • Woppmann A.
      • Will C.L.
      • Kiornstadt U.
      • Zuo P.
      • Manley J.L.
      • Luhrmann R.
      ,
      • Nikolakaki E.
      • Simos G.
      • Georgatos S.D.
      • Giannakouros T.
      ,
      • Nayler O.
      • Stamm S.
      • Ullrich A.
      ). Both hyper- and hypophosphorylation of SR proteins has been shown to influence splicing (
      • Okamoto Y.
      • Onogi H.
      • Honda R.
      • Yasuda H.
      • Wakabayashi T.
      • Nimura Y.
      • Hagiwara M.
      ,
      • Yeakley J.M.
      • Tronchere H.
      • Olesen J.
      • Dyck J.A.
      • Wang H.Y.
      • Fu X.D.
      ,
      • Prasad J.
      • Colwill K.
      • Pawson T.
      • Manley J.L.
      ), and the interaction of SR protein kinases with SR proteins can also influence their subcellular localization (
      • Koizumi J.
      • Okamoto Y.
      • Onogi H.
      • Mayeda A.
      • Krainer A.R.
      • Hagiwara M.
      ,

      Deleted in proof.

      ). However, at this time the regulation of SR protein kinases by peptide hormone-activated signal transduction pathways has not been demonstrated to our knowledge.
      The precise mechanisms by which SR proteins govern alternative splicing are under investigation in many laboratories. One model proposes that different concentrations of spliceosomal proteins in different cell types cause alternative processing of pre-mRNAs. Evidence for this mechanism is based on the variable expression levels of some SR proteins in tissues as a function of cell growth or differentiation (
      • Screaton G.R.
      • Caceres J.F.
      • Mayeda A.
      • Bell M.V.
      • Plebanski M.
      • Jackson D.G.
      • Bell J.I.
      • Krainer A.R.
      ,
      • Ayane M.
      • Preuss U.
      • Kohler G.
      • Nielsen P.J.
      ,
      • Mayeda A.
      • Krainer A.R.
      ,
      • Zahler A.M.
      • Neugebauer K.M.
      • Lane W.S.
      • Roth M.B.
      ). Another model proposes the existence of cell and/or developmental specific splicing factors that modulate splice site selection. For example, the female-specific expression ofDrosophila transformer protein determines the sexual fate of the fruit fly by directing splicing decisions (
      • Nayler O.
      • Schnorrer F.
      • Stamm S.
      • Ullrich A.
      ,
      • Du C.
      • McGuffin M.E.
      • Dauwalder B.
      • Rabinow L.
      • Mattox W.
      ). In addition, our recent finding that insulin regulated 5′-splice site selection of the PKCβII-specific exon within minutes after it binds to cell surface receptors suggested a third possibility. SR proteins could regulate alternative splicing via a receptor-linked signaling pathway responding to metabolic change rather than to a change in growth or development (
      • Chalfant C.E.
      • Watson J.E.
      • Bisnauth L.D.
      • Kang J.B.
      • Patel N.
      • Obeid L.M.
      • Eichler D.C.
      • Cooper D.R.
      ,
      • Chalfant C.E.
      • Mischak H.
      • Watson J.E.
      • Winkler B.C.
      • Goodnight J.
      • Farese R.V.
      • Cooper D.R.
      ).
      It is well known that insulin binding to its receptor activates at least three kinase pathways that can signal to the nucleus (
      • Czech M.P.
      • Corvera S.
      ,
      • Sawka-Verhelle D.
      • Filloux C.
      • Tartare-Deckert S.
      • Mothe I.
      • Van Obberghen E.
      ). Insulin-induced mitogen-activated protein kinase (MAPK) activation is associated with mitogenic signaling of insulin, and insulin-induced signal transducers and activators of transcription or JAK/signal transducers and activators of transcription pathways lead to nuclear transcriptional activator and repressor activation involved in cell differentiation (
      • Brunet A.
      • Bonni A.
      • Zigmond M.J.
      • Lin M.Z.
      • Juo P.
      • Hu L.S.
      • Anderson M.J.
      • Arden K.C.
      • Blenis J.
      • Greenberg M.E.
      ,
      • Sawka-Verhelle D.
      • Tartare-Deckert S.
      • Decaux J.F.
      • Girard J.
      • Van Obberghen E.
      ). In contrast, insulin activation of the phosphatidylinositol 3-kinase pathway is associated with metabolic signaling, consistent with the observation that insulin regulates PKCβII exon inclusion independent of cell growth and differentiation. Therefore, the possibility that a PI 3-kinase-dependent signaling pathway could alter the phosphorylation of post-transcriptional regulatory factors such as SR proteins as a step in the regulation of PKCβII expression was examined.
      Our studies focused initially on SRp40 for the following reasons. One, it was first described as an early response gene (HRS/SRp40). Two, SRp40 concentrations were increased by insulin in the regenerating liver where it is induced as a delayed early gene. Three, SRp40 levels are transcriptionally up-regulated by insulin. Four, SRp40 effects on exon inclusion have been demonstrated previously for the alternative splicing of fibronectin mRNA (
      • Du K.
      • Leu J.I.
      • Peng Y.
      • Taub R.
      ). Finally, increased SR protein concentrations during development, cell differentiation, and cell proliferation determine alternative splicing decisions (
      • Screaton G.R.
      • Caceres J.F.
      • Mayeda A.
      • Bell M.V.
      • Plebanski M.
      • Jackson D.G.
      • Bell J.I.
      • Krainer A.R.
      ,
      • Diamond R.H.
      • Du K.
      • Lee V.M.
      • Mohn K.L.
      • Haber B.A.
      • Tewar D.S.
      • Taub R.
      ,
      • Du K.
      • Peng Y.
      • Greenbaum L.E.
      • Haber B.A.
      • Taub R.
      ). In our case, however, insulin regulation of PKCβII exon inclusion in BC3H-1 myocytes and L6 myotubes occurs within 15 min, prior to SRp40 transcriptional up-regulation and increases in its concentration. This suggested that if SRp40 was involved in the insulin-induced alternative splicing that results in PKCβII mRNA, there must be another mechanism that influences SRp40 activity, other than changes in concentration.
      In the present study, we provide evidence to support SRp40 involvement in the regulation of PKCβII exon inclusion by insulin via its increased phosphorylation by a PI 3-kinase-dependent pathway.

      DISCUSSION

      Insulin is known to activate PI 3-kinase (
      • Czech M.P.
      • Corvera S.
      ), and several downstream kinases are activated in a PI 3-kinase-dependent manner including Akt, PKCζ, and PKCβII in skeletal muscle (
      • Chalfant C.E.
      • Ohno S.
      • Konno Y.
      • Fisher A.A.
      • Bisnauth L.
      • Watson J.E.
      • Cooper D.R.
      ,
      • Braiman L.
      • Sheffi-Friedman L.
      • Bak A.
      • Tennenbaum T.
      • Sampson S.R.
      ,
      • Cooper D.R.
      • Watson J.E.
      • Patel N.
      • Illingworth P.
      • Duncan-Acevedo M.
      • Goodnight J.
      • Chalfant C.E.
      • Mischak H.
      ,
      • Thorell A.
      • Hirshman M.F.
      • Nygren J.
      • Jorfeldt L.
      • Wojtaszewski J.F.
      • Dufresne S.D.
      • Horton E.S.
      • Ljungqvist O.
      • Goodyear L.J.
      ). Insulin signaling to the nucleus by PI 3-kinase is not as well studied, but it is associated with activation of gene transcription in addition to its roles in glycogen and protein synthesis and glucose transport (
      • Withers D.J.
      • White M.
      ). Our study found increased phosphorylation of SR proteins by insulin-dependent PI 3-kinase pathways. This indicated that factors involved in 5′-splice site selection could be regulated by insulin signaling mechanisms.
      The C-terminal portion of SR proteins contains a domain rich in serine and arginine residues that is highly phosphorylated. At least eight members of the SR family, including SRp40 and SF2/ASF, contain phosphoepitopes that are recognized by mAb104 (
      • Roth M.B.
      • Zahler A.M.
      • Stolk J.A.
      ). The finding that insulin treatment increased the phosphorylation state of at least seven proteins in nuclear extracts from skeletal muscle cells is consistent with the observation that the RS domains are highly phosphorylatedin vivo (
      • Gui J.-F.
      • Lane W.S.
      • Fu X.-D.
      ,
      • Colwill K.
      • Pawson T.
      • Andrews B.
      • Prasad J.
      • Manley J.L.
      • Bell J.C.
      • Duncan P.I.
      ).
      We focused on HRS/SRp40, a splicing factor with a molecular mass of about 40 kDa that was originally shown to be induced by insulin in rat hepatocytes (
      • Feramisco J.R.
      • Burridge K.
      ). By using immunoprecipitation of SRp40 followed by detection with an antibody to examine serine phosphorylation specifically, it became evident that insulin treatment increased the phosphorylation state of the SR protein rather than its concentration. The phosphorylation was blocked by pretreatment with LY294002 as was splicing. This indicated a role for PI 3-kinase in alternative splicing, and to our knowledge, this is the first report of a hormone signaling the phosphorylation of an SR protein.
      Since the overexpression of trans-factors has also been useful in establishing their role in splicing, SRp40 cDNA was expressed in differentiated myotubes and resulted in exon inclusion. This was used as evidence to link the regulation of SR concentration to splice site selection in previous studies (
      • Du K.
      • Peng Y.
      • Greenbaum L.E.
      • Haber B.A.
      • Taub R.
      ). Here, the overexpression was linked to increased RS domain phosphorylation, increased PKCβII mRNA, increased PKCβII protein, and to increased glucose transport. It is premature to suggest which downstream kinases or phosphatases are activated or inhibited by PI 3-kinase to result in increased SR protein phosphorylation. It is possible that insulin-activated kinases such as Akt or PKC could phosphorylate SR proteins (
      • Obata T.
      • Yaffe M.B.
      • Leparc G.G.
      • Piro E.T.
      • Maegawa H.
      • Kashiwagi A.
      • Kikkawa R.
      • Cantley L.C.
      ).
      Two consensus sequences have been proposed for SRp40-binding sites (
      • Tacke R.
      • Chen Y.
      • Manley J.L.
      ,
      • Liu H.-X.
      • Zhang M.
      • Krainer A.R.
      ). Both are present in the introns flanking the βII-specific exon. The first sequence occurs prior to the 3′-pyrimidine tract (ACDGS). The second sequence we identified by sequence analysis is longer, occurs about 350 bp after the first 5′-splice site, and corresponds closely to one described (
      • Tacke R.
      • Chen Y.
      • Manley J.L.
      ). When the second site was targeted using antisense oligonucleotides, exon inclusion was inhibited. The use of antisense oligonucleotides for down-regulating gene expression is well documented where sequences are targeted to block translation or lead to destabilization of the message by RNase H or inhibit transcription by forming triplex structures within the promoter regions of DNA. Antisense oligonucleotides have also been used to restore splicing of mutated pre-mRNA in thalassemic β-globin and to redirect splice site selection for Bcl-xS versus Bcl-xL independent of down-regulating gene expression (
      • Taylor J.K.
      • Dean N.M.
      ,
      • Taylor J.T.
      • Zhang Q.Q.
      • Wyatt J.R.
      • Dean N.M.
      ,
      • Gormann L.
      • Mercatante D.R.
      • Kole R.
      ). Here, blocking one site for SRp40-RNA interaction provided mutual dependence of a downstream sequence with insulin-induced changes in SRp40 phosphorylation. This finding is analogous to studies where antisense toward SF2/ASF-binding sites blocked splicing of bGH pre-mRNA in vitro(
      • Dirksen W.P.
      • Mayeda A.
      • Krainer A.R.
      • Rottman F.M.
      ).
      The effect of the PI 3-kinase inhibitor, LY294002, to block glucose uptake has been reported (
      • Bandyopadhyay G.
      • Standaert M.L.
      • Galloway L.
      • Moscat J.
      • Farese R.V.
      ), and its effect is consistent with a role for PI 3-kinase in insulin action (
      • Pessin J.E.
      • Saltiel A.R.
      ) since this signaling pathway directly links downstream kinases with the recruitment of glucose carriers to the plasma membrane and results in increased glucose uptake. Next, we evaluated the effect of newly synthesized PKCβII on insulin-stimulated glucose uptake. Cycloheximide blocked the recruitment of glucose carriers in adipose cells (
      • Baly D.L.
      • Horuk R.
      ,
      • Matthaei S.
      • Olefsky J.M.
      • Karnieli E.
      ). Although there is a conflicting report in adipocytes (
      • Jones T.L.
      • Cushman S.W.
      ), differences in the preparation and pretreatment of cells could be involved. In this study, cycloheximide treatment blocked insulin effects in serum-depleted myotubes.
      We demonstrated that overexpression of SRp40 mimicked insulin to increase basal glucose uptake. This is consistent with the effects of increased PKCβII concentrations that occur following SRp40 overexpression. Since increased SRp40 concentrations may be altering alternative splicing of other pre-mRNA in a nonspecific manner, this correlation should be interpreted tentatively. For example, the insulin receptor is also alternatively spliced, and the B form of the receptor is thought to signal more effectively (
      • Kosaki A.
      • Webster N.J.G.
      ). However, the ability of antisense oligonucleotides targeting the SRp40-binding site to block insulin effects on glucose transport suggests that the alteration in splicing alone is responsible for the increase in transport. Finally, LY379196, a PKCβ inhibitor which blocks glucose uptake in primary mouse myotubes (
      • Braiman L.
      • Sheffi-Friedman L.
      • Bak A.
      • Tennenbaum T.
      • Sampson S.R.
      ), also inhibited insulin effects on glucose uptake.
      Our studies in cells support in vitro observations for an SRp40 role in splice site selection where addition of one or more SR proteins to in vitro deficient splicing extracts restored splice site selection in a concentration-dependent manner (
      • Stark J.M.
      • Cooper T.A.
      • Roth M.B.
      ,
      • Stark J.M.
      • Bazet-Jones D.P.
      • Herfort M.
      • Roth M.B.
      ). The identification of SRp40 as a component of insulin-regulated splicing was defined by the following criteria: its ability to mimic insulin effects on PKCβII splicing, inhibition of its phosphorylation state by LY294002, a compound that blocks insulin activation of PI 3-kinase, and the ability of 2′-O-methoxyethyl antisense oligonucleotides directed to a putative SRp40 site to block insulin effects on splicing as well as to block insulin effects on glucose transport. The demonstration of SRp40 as a factor regulated by a PI 3-kinase signaling cascade provides an additional mechanism for regulating alternative splicing. SRp40 phosphorylation correlates to alternative splicing of the βII exon in a manner analogous to insulin treatment and links a signaling pathway to exon inclusion events in vivo. Unlike systems of tissue-specific alternative splicing, however, the concentration of SRp40 did not change with insulin treatment, rather its phosphorylation state increased. Taken together, the multiple strategies used here to investigate SRp40 interactions in intact cells indicate a pivotal role for this trans-factor and PI 3-kinase in insulin-stimulated alternative splicing of PKCβ pre-mRNA and subsequent effects of insulin on glucose transport.

      Acknowledgments

      We thank Dr. Rebecca Taub, Department of Genetics, University of Pennsylvania School of Medicine, for generously providing pCMV-SRp40 and polyclonal SRp40 antibody. We thank Dr. James L. Manley, Department of Biological Sciences, Columbia University, for discussions. We also thank Konrad Mebert, Dan Mancu, and David Chappell for excellent technical assistance.

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