Abstract 3884: Beta-2 spectrin opposes the CDK4-mediated suppression of TGF-beta signaling by rescuing Smad3 nuclear localization

Abstract

Abstract Background: Transforming growth factor beta (TGF-β) signaling inhibits cell proliferation by inducing a G1 cell cycle arrest. By contrast, cyclin-dependent kinase 4 (CDK4) inactivates retinoblastoma function early in G1, thus promoting cell cycle progression. However, CDK4 can also inhibit TGF-β signaling by phosphorylating the linker region of Smad3, a key mediator of TGF-β signaling. We have previously demonstrated that Smad3 nuclear translocation and TGF-β signaling are dependent upon the function of the adaptor protein, beta-2 spectrin (β2SP or ELF). Furthermore, loss of β2SP in a knockout model results in the spontaneous development of hepatocellular cancer (HCC). HCCs from these elf+/− mice overexpress CDK4, and haploinsufficiency of CDK4 in elf+/−/CDK4neo/+ mice suppresses hepatocarcinogenesis. These findings point towards competing activities of β2SP and CDK4 on TGF-β signaling, which may be determined by their modulation of Smad3. Our goal was to determine the mechanisms for these competing effects. Methods: We have overexpressed CDK4, β2SP, or both in human HCC cell lines. Using these stable cell lines, we examined Smad3 phosphorylation by western blot, Smad3 nuclear localization by immunocytochemistry, and TGF-β-regulated gene expression by quantitative RT-PCR (qPCR). Results: We sought to determine whether β2SP can rescue the CDK4-mediated suppression of TGF-β signaling. Over-expression of CDK4 blocked TGF-β-induced Smad3 nuclear translocation, whereas a CDK4/6-specific inhibitor, PD-0332991 (Pfizer, Inc) rescued Smad3 nuclear translocation. Similarly, over-expression of β2SP rescued Smad3 nuclear translocation in these CDK4 overexpressing cells. Furthermore, both PD-0332991 and β2SP overexpression enhanced the expression of TGF-β-regulated genes, as determined by qPCR. We hypothesize that the opposing effects of CDK4 and β2SP are mediated by the balance between the CDK4-mediated phosphorylation of the Smad3 linker region, and the β2SP-mediated phosphorylation of the Smad3 C-terminal domain. Thus, we also examined the domain-specific phosphorylation of Smad3 in CDK4-overexpressing cells. PD-0332991 specifically reduces phosphorylation of the linker region of Smad3 (T179 and S208), but has no effect on c-terminal phosphorylation (S423/S425), while overexpression of β2SP increases c-terminal phosphorylation. Assessment of Smad3 nuclear localization and the effects on TGF-β signaling in the presence of phospho-specific Smad3 mutants is ongoing. Conclusions: Our results have begun to clarify the competing roles of CDK4 and β2SP on Smad3 nuclear localization and thus TGF-β signaling. Based on our results, targeted-inhibition of the CDK4-Smad3 interaction may be critical in rescuing TGF-β-mediated regulation of cell cycle control, but β2SP expression may be required, and may serve as a critical marker predictive of such activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3884.