Smad7 and Smurf2 Ub Ligase are Co-operative Partners in Regulating TGF-β Signaling

RiboEvo Special Seminar Series

Ubiquitylation targets proteins for destruction and controls both protein cellular function and levels. This posttranslational modification of proteins with ubiquitin (Ub) involves concerted activities of three enzymes, namely Ub-activating (E1), Ub-conjugating (E2) and Ub-ligating (E3) enzymes. The regulation of enzymes implicated in the ubiquitylation process is crucial to determining their roles in cellular processes. The TGFβ superfamily of cytokines regulates growth and differentiation via signaling cascades initiated from heteromeric TGFβ receptor complexes and propagated by the Smad proteins. One aspect of signal attenuation is an ubiquitin-dependent process that requires the recruitment of Smurf2, a C2-WW-HECT containing E3-ubiquitin ligase by the receptor-inhibitory Smad, Smad7. Although the C-terminal MH2 domain of Smad7 has been shown to be required for receptor association, the amino-terminal domain (NTD) remains poorly characterized. The amino-terminal domain of Smad7 is here demonstrated to be required to stimulate the catalytic activity of the Smurf2 HECT domain by recruiting the E2, UbcH7 to the HECT domain. A 2.1Å resolution x-ray crystal structure of the Smurf2 HECT domain reveals that it has a sub-optimal E2 binding pocket that could be optimized by mutagenesis to generate a HECT domain that functions independently of Smad7 and potently inhibits TGFβ signaling. Thus, a sub-optimal E2 binding groove in Smurf2 HECT domain confers a requirement for an adaptor protein to efficiently associate with its cognate E2. Therefore, E2 enzyme recognition by an E3 HECT enzyme is not constitutively competent and provides a point of control for regulating the ubiquitin ligase catalytic activity via the action of an auxillary protein.