KRAS Protein Stability Is Regulated through SMURF2: UBCH5 Complex-Mediated β-TrCP1 Degradation

作者：Shirish Shukla, Uday Sankar Allam, Aarif Ahsan, Guoan Chen, Pranathi Meda Krishnamurthy, Katherine B. Marsh, Matthew Rumschlag, Sunita Shankar, Christopher E. Whitehead, Matthew J. Schipper, Venkatesha Basrur, Daniel R. Southworth, Arul M. Chinnaiyan, Alnawaz Rehemtulla, David G. Beer, Theodore Steven Lawrence, Mukesh Kumar Nyati, Dipankar Ray · 发表于：Neoplasia · 年份：2014 · DOI：10.1593/neo.14184 · 被引用次数：101 · 研究领域：Ubiquitin and proteasome pathways、Cancer-related Molecular Pathways、Protein Degradation and Inhibitors

Attempts to target mutant KRAS have been unsuccessful. Here, we report the identification of Smad ubiquitination regulatory factor 2 (SMURF2) and UBCH5 as a critical E3:E2 complex maintaining KRAS protein stability. Loss of SMURF2 either by small interfering RNA/short hairpin RNA (siRNA/shRNA) or by overexpression of a catalytically inactive mutant causes KRAS degradation, whereas overexpression of wild-type SMURF2 enhances KRAS stability. Importantly, mutant KRAS is more susceptible to SMURF2 loss where protein half-life decreases from >12 hours in control siRNA-treated cells to <3 hours on Smurf2 silencing, whereas only marginal differences were noted for wild-type protein. This loss of mutant KRAS could be rescued by overexpressing a siRNA-resistant wild-type SMURF2. Our data further show that SMURF2 monoubiquitinates UBCH5 at lysine 144 to form an active complex required for efficient degradation of a RAS-family E3, β-transducing repeat containing protein 1 (β-TrCP1). Conversely, β-TrCP1 is accumulated on SMURF2 loss, leading to increased KRAS degradation. Therefore, as expected, β-TrCP1 knockdown following Smurf2 siRNA treatment rescues mutant KRAS loss. Further, we identify two conserved proline (P) residues in UBCH5 critical for SMURF2 interaction; mutation of either of these P to alanine also destabilizes KRAS. As a proof of principle, we demonstrate that Smurf2 silencing reduces the clonogenic survival in vitro and prolongs tumor latency in vivo in cancer cells inclu...