Abstract
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.
Highlights
The pro-proliferative Krüppel-like factor 5 (KLF5) is posttranslationally regulated
When HA-tagged KLF5 and Myc-tagged SMAD ubiquitination regulatory factor 2 (SMURF2) were cotransfected in COS-1 cells stabilized with MG132, a proteasome-specific inhibitor, immunoprecipitation with a HA antibody followed by Western blotting against Myc indicated that Myc-SMURF2 coimmunoprecipitated with HA-KLF5 (Fig. 1C)
We demonstrate a novel interaction between KLF5 and SMURF2
Summary
Results: SMAD ubiquitination regulatory factor 2 (SMURF2) interacts with, ubiquitinates and degrades KLF5. SMURF2 is a novel E3 ubiquitin ligase for KLF5 and negatively regulates KLF5 by targeting it for proteasomal degradation. SMURF2 is an E3 ubiquitin ligase recently grouped into the Nedd family of HECT ubiquitin ligases [2] It contains WW domains, which directly bind to a PPXY motif ( known as PY motif) in its targets [2]. As a HECT E3 ubiquitin ligase, SMURF2 catalyzes ubiquitination at specific lysine residues in its targets, which triggers subsequent degradation by proteasomes [2]. A primary mechanism by which KLF5 is posttranslationally regulated is through ubiquitination and subsequent degradation of KLF5, as mediated by a number of E3 ubiquitin ligases [5, 7, 17, 19], including WWP1 and FBW7 [5, 7, 19]. This report presents KLF5 as a target for SMURF2 and SMURF2 as an ubiquitin ligase that regulates KLF5
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