Protein Kinase C-Dependent Control of Bcl-x Alternative Splicing

Abstract

The alternative splicing of Bcl-x generates the proapoptotic Bcl-x S protein and the antiapoptotic isoform Bcl-x L . Bcl-x splicing is coupled to signal transduction, since ceramide, hormones, and growth factors alter the ratio of the Bcl-x isoforms in different cell lines. Here we report that the protein kinase C (PKC) inhibitor and apoptotic inducer staurosporine switches the production of Bcl-x towards the x S mRNA isoform in 293 cells. The increase in Bcl-x S elicited by staurosporine likely involves signaling events that affect splicing decisions, because it requires active transcription and no new protein synthesis and is independent of caspase activation. Moreover, the increase in Bcl-x S is reproduced with more specific inhibitors of PKC. Alternative splicing of the receptor tyrosine kinase gene Axl is similarly affected by staurosporine in 293 cells. In contrast to the case for 293 cells, PKC inhibitors do not influence the alternative splicing of Bcl-x and Axl in cancer cell lines, suggesting that these cells have sustained alterations that uncouple splicing decisions from PKC-dependent signaling. Using minigenes, we show that an exonic region located upstream of the Bcl-x S 5 splice site is important to mediate the staurosporine shift in Bcl-x splicing. When transplanted to other alternative splicing units, portions of this region confer splicing modulation and responsiveness to staurosporine, suggesting the existence of factors that couple splicing decisions with PKC signaling.Alternative splicing of pre-mRNAs provides a powerful mechanism to augment the protein repertoire encoded by metazoan genomes. It is estimated that 74% of all multiexonic human genes are alternatively spliced (36). Moreover, alternative splicing is becoming increasingly relevant to a variety of human diseases, including cancer (60, 85). These observations justify current efforts devoted at uncovering the basic principles of alternative splicing control. Many studies have provided valuable insights into the roles of specific elements and factors in splicing modulation (7,58,84). Proteins that bind to specific sequence elements to affect splice site selection include SR proteins, hnRNP proteins, and other related RNA binding proteins such as TIA-1, ETR-3, and Sam68 (16,29,35,54,59,76). Many of these proteins can be modified posttranslationally, and some of the modifications, like phosphorylation, can affect their localization and activity (59, 82).Although we expect that the coupling between signal transduction events and alternative splicing decisions will represent a major network of regulation, very little is known about how splicing is coordinated by a variety of effectors to attune cells to specific environmental demands and stresses. Possibly the first system describing a link between signal transduction and splicing involved the cell surface molecule CD44, in which exon v5 inclusion was stimulated following activation of the protein kinase C (PKC) or extracellular signal-regulated kinase, as part of the Ras signaling path...

TCERG1 Regulates Alternative Splicing of the Bcl-x Gene by Modulating the Rate of RNA Polymerase II Transcription

“…It has been reported that a cis regulatory element, called SB1, is located in the first half of Bcl-x exon 2 (55). This 361-nucleotide-long region behaves as a splicing silencer, and its deletion stimulates the use of the Bcl-x S 5= splice site, thus promoting the expression of the Bcl-x S isoform in transfection assays performed with HEK293 and HeLa cells (55). We sought to determine whether TCERG1 affects Bcl-x alternative splicing through the SB1 element.…”

Multiple and Specific mRNA Processing Targets for the Major Human hnRNP Proteins

“…The same blot was decorated with an anti-actin antibody (Sigma A2066) to correct for total protein content in different lanes. Anti-hnRNP A1 and A2 antibodies and anti-hnRNP F and H antibodies were described previously (49,50). Anti-hnRNP C1/C2 and G (C-17) were from Santa Cruz Biotechnologies, Inc. (sc-10037 and sc-4583, respectively); anti-hnRNP D/AUF1 was purchased from Upstate Biotechnology, Inc. (catalog no.…”

Reactivation of Fetal Splicing Programs in Diabetic Hearts Is Mediated by Protein Kinase C Signaling

“…PKC␣/␤ has been previously linked to AS 4 regulation (11)(12)(13) and identified as one of the regulators of a group of AS events in HEK293T cells activated by epidermal growth factor (14). AS regulates eukaryotic gene expression by controlling the inclusion and exclusion of alternative exons within the coding and noncoding RNA transcripts (15,16).…”