KRAS protein stability is regulated through SMURF2: UBCH5 complex-mediated β-TrCP1 degradation.
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 including mutant KRAS-driven tumors. Taken together, we show that SMURF2:UBCH5 complex is critical in maintaining KRAS protein stability and propose that targeting such complex may be a unique strategy to degrade mutant KRAS to kill cancer cells.
Department of Radiation Oncology, University of Michigan, Ann Arbor, MI.
Shirish
S
Shukla
Department of Radiation Oncology, University of Michigan, Ann Arbor, MI.
Uday Sankar
US
Allam
Department of Radiation Oncology, University of Michigan, Ann Arbor, MI.
Aarif
A
Ahsan
Department of Surgery, University of Michigan, Ann Arbor, MI.
Guoan
G
Chen
Department of Radiation Oncology, University of Michigan, Ann Arbor, MI.
Pranathi Meda
PM
Krishnamurthy
Department of Radiation Oncology, University of Michigan, Ann Arbor, MI.
Katherine
K
Marsh
Department of Radiation Oncology, University of Michigan, Ann Arbor, MI.
Matthew
M
Rumschlag
Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, MI.
Sunita
S
Shankar
Department of Radiology, University of Michigan, Ann Arbor, MI.
Christopher
C
Whitehead
Department of Radiation Oncology, University of Michigan, Ann Arbor, MI.
Matthew
M
Schipper
Department of Pathology, University of Michigan, Ann Arbor, MI.
Venkatesha
V
Basrur
Department of Biological Chemistry, University of Michigan, Ann Arbor, MI.
Daniel R
DR
Southworth
Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, MI; Department of Pathology, University of Michigan, Ann Arbor, MI.
Arul M
AM
Chinnaiyan
Department of Radiation Oncology, University of Michigan, Ann Arbor, MI.
Alnawaz
A
Rehemtulla
Department of Surgery, University of Michigan, Ann Arbor, MI.
David G
DG
Beer
Department of Radiation Oncology, University of Michigan, Ann Arbor, MI.
Theodore S
TS
Lawrence
Department of Radiation Oncology, University of Michigan, Ann Arbor, MI.
Mukesh K
MK
Nyati
Department of Radiation Oncology, University of Michigan, Ann Arbor, MI. Electronic address: dipray@umich.edu.
Dipankar
D
Ray
D000230:Adenocarcinoma; D000077192:Adenocarcinoma of Lung; D020816:Amino Acid Motifs; D000818:Animals; D005260:Female; D015870:Gene Expression; D045325:HCT116 Cells; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008175:Lung Neoplasms; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D011485:Protein Binding; D055550:Protein Stability; D059748:Proteolysis; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras); D044763:Ubiquitin-Conjugating Enzymes / Q000502:physiology*; D044767:Ubiquitin-Protein Ligases / Q000502:physiology*; D054875:Ubiquitination; D044844:beta-Transducin Repeat-Containing Proteins / Q000502:physiology*; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
C471644:BTRC protein, human; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D044844:beta-Transducin Repeat-Containing Proteins; C097308:UBE2D1 protein, human; D044763:Ubiquitin-Conjugating Enzymes; C471641:SMURF2 protein, human; D044767:Ubiquitin-Protein Ligases; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins