The zinc finger transcription factor Krüppel-like factor 5 (KLF5) is regulated 
posttranslationally. We identified SMAD ubiquitination regulatory factor 2 
(SMURF2), an E3 ubiquitin ligase, as an interacting protein of KLF5 by yeast 
two-hybrid screen, coimmunoprecipitation, and indirect immunofluorescence 
studies. The SMURF2-interacting domains in KLF5 were mapped to its carboxyl 
terminus, including the PY motif of KLF5 and its zinc finger DNA-binding domain. 
KLF5 protein levels were reduced significantly upon overexpression of SMURF2 but 
not catalytically inactive SMURF2-C716A mutant or SMURF1. SMURF2 alone reduced 
the protein stability of KLF5 as shown by cycloheximide chase assay, indicating 
that SMURF2 specifically destabilizes KLF5. In contrast, KLF5(1-165), a KLF5 
amino-terminal construct that lacks the PY motif and DNA binding domain, was not 
degraded by SMURF2. The degradation of KLF5 by SMURF2 was blocked by the 
proteasome inhibitor MG132, and SMURF2 efficiently ubiquitinated both 
overexpressed and endogenous KLF5. In contrast, knocking down SMURF2 by siRNAs 
significantly enhanced KLF5 protein levels, reduced ubiquitination of KLF5, and 
increased the expression of cyclin D1 and PDGF-A, two established KLF5 target 
genes. In consistence, SMURF2, but not the E3 ligase mutant SMURF2-C716A, 
significantly inhibited the transcriptional activity of KLF5, as demonstrated by 
dual luciferase assay using the PDGF-A promoter, and suppressed the ability of 
KLF5 to stimulate cell proliferation as measured by BrdU incorporation. Hence, 
SMURF2 is a novel E3 ubiquitin ligase for KLF5 and negatively regulates KLF5 by 
targeting it for proteasomal degradation.