Abstract 5892: Degradation and Intranuclear Redistribution of Wilms’ Tumor 1-Associating Protein Orchestrate Survivin Splice Variant Switching and Underlie IGF-1-Mediated Vascular Smooth Muscle Cell Survival
We recently discovered that Wilms’ tumor 1-associating protein (WTAP), an essential nuclear protein, was dynamically expressed in the artery wall and can be pro-apoptotic for human vascular smooth muscle cells (SMCs, Circ Res, 2006). The Drosophila homolog of WTAP regulates pre-mRNA splicing however the mechanism of WTAP action in mammalian cells is uncertain. To identify a regulatory cascade for WTAP action, we screened growth and survival factors for their effects on WTAP expression and found that insulin-like growth factor-1 (IGF-1) uniquely stimulated a rapid (12 hour) decline in WTAP abundance. This decline in WTAP was necessary for IGF-1 to confer its anti-apoptotic properties, which were blocked by transducing the WTAP gene into SMCs. We further found that WTAP downregulation by IGF-1 was mediated by a PI3K-Akt signaling axis, as indicated by both pharmacological and shRNA strategies, and that this cascade directed the degradation of WTAP via a nuclear 26S proteasome. Furthermore, we determined that upon WTAP degradation, the pre-mRNA splicing program for the survival factor, survivin, was strikingly shifted with reduced expression of survivin-2B, which is pro-apoptotic, and increase expression of survivin, which is anti-apoptotic. Knockdown of survivin-2B rescued the ability of IGF-1 to promote survival, assessed by Annexin-V binding, when WTAP was overexpressed. We also identified ATM phosphorylation sites on WTAP that were responsible for a rapid (10 min), IGF-1-mediated release of WTAP from the spliceosome, as assessed by nuclear extract gel filtration. Site-directed mutagenesis of ATM target sites prevented IGF-1-mediated release from the spliceosome and skewed the survivin splicing output toward survivin-2B. Conclusion: These data uncover a novel regulatory cascade for human SMC survival based on adjusting the nuclear abundance and spliceosome association of WTAP. These changes, in turn, define the splice variant balance between pro-and anti-apoptotic isoforms of survivin.