title: The E3 ubiquitin ligase SMAD ubiquitination regulatory factor 2 negatively regulates Krüppel-like factor 5 protein.
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.
authors.affiliation: Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
authors.forename: James X
authors.initials: JX
authors.lastname: Du
authors.forename: Engda G
authors.initials: EG
authors.lastname: Hagos
authors.forename: Mandayam O
authors.initials: MO
authors.lastname: Nandan
authors.forename: Agnieszka B
authors.initials: AB
authors.lastname: Bialkowska
authors.forename: Bing
authors.initials: B
authors.lastname: Yu
authors.forename: Vincent W
authors.initials: VW
authors.lastname: Yang
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D019938:Cyclin D1 / Q000096:biosynthesis / Q000235:genetics; D005786:Gene Expression Regulation / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D010982:Platelet-Derived Growth Factor / Q000096:biosynthesis / Q000235:genetics; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology*
chemical_list: C530104:CCND1 protein, human; C492214:KLF5 protein, human; D051741:Kruppel-Like Transcription Factors; D010982:Platelet-Derived Growth Factor; C075278:platelet-derived growth factor A; D019938:Cyclin D1; C471641:SMURF2 protein, human; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex