Tumor suppressor Smad4/DPC4 is a central intracellular signal transducer for transforming growth factor-␤ (TGF-␤) signaling. We recently reported that transcriptional potential of Smad4 was regulated by SUMOylation in transfected HeLa cells (1), but the precise mechanism and function of Smad4 SUMOylation in TGF-␤ signaling remain to be elucidated. Here, we describe the regulation of TGF-␤ signaling by SUMOylation through the control of Smad4 metabolic stability and subcellular localization. We

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... ound that SUMO-1 overexpression strongly increases Smad4 levels, while inhibition of SUMOylation by small interfering RNA (siRNA)-mediated knockdown of the E2 enzyme Ubc9 reduces endogenous Smad4 levels. Concomitantly, SUMO-1 overexpression enhances and Ubc9 knockdown reduces levels of intranuclear Smad4, growth inhibitory response, as well as transcriptional responses to TGF-␤. Comparison of wild type and mutant forms of Smad4 for SUMOylation, ubiquitination, and half-life allows the conclusion that SUMO-1 modification serves to protect Smad4 from ubiquitin-dependent degradation and consequently enhances the growth inhibitory and transcriptional responses of Smad4.