Identification of substrates of SMURF1 ubiquitin ligase activity utilizing protein microarrays

The ubiquitin proteasome pathway (UPP) has been implicated in a number of pathogenic diseases: cancer, inflammation, metabolic disorders, and viral infection. The human genome contains well over 500 genes encoding proteins involved in the UPP. Ubiquitin ligases (E3s) comprise the largest subset of these genes, and together with an E2 partner, provide the substrate selectivity required for regulating cellular proteins through the covalent attachment of ubiquitin. Many ligases that have been identified in critical cellular pathways have no known substrates. Even those E3s with known substrates may have a yet unidentified role in the pathways on which they lie and as such may have additional substrates. It is critical to identify these substrates for discovery of selective small molecule inhibitors aimed at therapeutic intervention. Other methods, such as mass spectrometry, have been utilized for identifying ligase substrates, but these are labor-intensive and require a significant investment. In this study, we utilized protein microarrays for the identification of substrates of the HECT domain E3, Smurf1. Smurf1 is a critical regulator of TGF-beta and bone morphogenic protein signaling, and has been demonstrated to play a role in regulating cell polarity through the degradation of RhoA. We set out to identify novel Smurf1 substrates involved in the regulation of the aforementioned pathways. Proof-of-principle experiments with known Smurf1 substrates demonstrated efficient ubiquitination thereby validating this approach. Assaying a human protein microarray for ubiquitination with Smurf1 and the partner E2 ubiquitin ligase Ubch5 or Ubch7 identified 89 potential substrates of the Smurf1 E3 activity, which spanned a number of different biological pathways. Substrates identified utilizing protein microarray technology have been validated in vitro. Here we demonstrate the utility of this approach for identifying substrates of particular E2/E3 complexes.