Recombinant Human SMURF2, GST-tagged

• Cat.No.: SMURF2-138H
• Product Overview: Recombinant human SMURF2 (amino acid residues 1-748), with N-terminal GST, was expressed in E.coli.

Specification

• Species: Human
• Source: E.coli
• Tag: GST
• Protein Length: 1-748 a.a.
• Description: The enzymes of the ubiquitylation pathway play a pivotal role in a number of cellular processes including the regulated and targeted proteasome-dependent degradation of substrate proteins. Three classes of enzymes are involved in the process of ubiquitylation; activating enzymes (E1s), conjugating enzymes (E2s) and protein ligases (E3s). Smad-Specific E3 Ubiquitin Protein Ligase 1 (SMURF2) is a member of the E3 protein ligase family and cloning of the human gene was first described by Kavsak et al. (2000). SMURF2 is a HECT domain ubiquitin E3 ligase that has been shown to regulate cell polarity, senescence and tumor suppression (Blank et al., 2012).
• Form: 50 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM dithiothreitol, 10% glycerol
• Molecular Mass: ~114kDa
• Storage: 12 months at -70°C. Avoid multiple freeze/thaw cycles.
• Concentration: 0.5mg/ml

Gene Information

• Gene Name: SMURF2 SMAD specific E3 ubiquitin protein ligase 2 [ Homo sapiens ]
• Official Symbol: SMURF2
• Synonyms: SMURF2; SMAD specific E3 ubiquitin protein ligase 2; E3 ubiquitin-protein ligase SMURF2; hSMURF2; E3 ubiquitin ligase SMURF2; SMAD ubiquitination regulatory factor 2; SMAD-specific E3 ubiquitin-protein ligase 2; MGC138150; DKFZp686F0270
• Gene ID: 64750
• mRNA Refseq: NM_022739
• Protein Refseq: NP_073576
• MIM: 605532
• UniProt ID: Q9HAU4
• Chromosome Location: 17q22-q23
• Pathway: Adaptive Immune System, organism-specific biosystem; Antigen processing: Ubiquitination and Proteasome degradation, organism-specific biosystem; BMP receptor signaling, organism-specific biosystem; Class I MHC mediated antigen processing & presentation, organism-specific biosystem; Endocytosis, organism-specific biosystem
• Function: SMAD binding; acid-amino acid ligase activity; identical protein binding; ligase activity; protein binding; ubiquitin-protein ligase activity

Citation

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

Journal: Neoplasia (New York, N.Y.)    PubMed ID: 24709419    Data: 2014/2/1

Authors: Shirish Shukla, Uday SankarAllam, Dipankar Ray

Article Snippet:The in vitro ubiquitination reaction was carried out in a 15-μl reaction volume containing reaction buffer [250 mM Tris-HCl (pH 7.5), 50 mM MgCl 2 , 50 μM DTT, and 20 mM ATP), 10 βg of Myc-tagged ubiquitin (Cat. No. U-115), 0.35 βg of UBE1 (Cat. No. E305), and 0.5 βg of UBCH5 (Cat. No. E2-616; all from Boston Biochemicals), and Flag-tagged β-TrCP1 was overexpressed in HEK293 cells and pulled down using affi-Flag (M2) beads.(pH 7.5), 50 mM MgCl 2 , 50 μM DTT, and 20 mM ATP), 10 βg of Myc-tagged ubiquitin (Cat. No. U-115), 0.35 βg of UBE1 (Cat. No. E305), and 0.5 βg of UBCH5 (Cat. No. E2-616; all from Boston Biochemicals), and Flag-tagged β-TrCP1 was overexpressed in HEK293 cells and pulled down using affi-Flag (M2) beads. ... Human recombinant SMURF2 protein (Cat. 468H; Creative BioMart, New York, NY) was then added, and the reaction mixtures were incubated at 37°C for 2 hours.. The reaction was terminated after boiling with 4x gel loading dye.The reaction was terminated after boiling with 4x gel loading dye.

SMURF2 ubiquitin ligase activity controls mutant KRAS steady-state levels. (A) H358 (KRASG12C), H441 (KRASG12V), and H2347 (KRASWT) human lung adenocarcinoma cells were transfected with either control (C) or Smurf2 (S) siRNA. Forty-eight hours posttransfection, cell lysates were subjected to immunoblot analysis using specified antibodies. (B) Quantification of KRAS steady-state level in H358, H441, and H2347 cells on C or S siRNA. Immunoblot obtained from A were scanned and quantified using ImageJ software, and KRAS steady-state levels were normalized using GAPDH as loading control. Relative KRAS protein levels were obtained, and mean ± SEM values were calculated from three independent experiments; * denotes significant difference from control at P < .05; **, denotes significant difference from control at P < .005; NS, not significant. (C) H441 cells were transfected with Smurf2 si-R (SM2-siR) construct before siRNA-mediated Smurf2 knockdown. Cell lysates were prepared 24 hours post siRNA transfection and immunoblotted using indicated antibodies. (D) HEK293 cells were co-transfected with Myc-tagged KRAS [either wild type or various mutants (G12D, V, C, or S)] in the presence or absence of FLAG-tagged SMURF2 [either wild type or C716A (CA) mutants]. Twelve hours post-transfection, cell lysates were prepared and subjected to immunoblot analysis using indicated antibodies.

Ubiquitin ligase activity of SMURF2 positively regulates KRAS protein stability. (A) HEK293 cells were transfected with KRAS in the presence or absence of SMURF2 (SM2) CA. Twelve hours post-transfection, indicated cells were treated with either 5 mM 3-MA or 2 μM MG132 for 4 hours. Cell lysates were then subjected to immunoblot analysis with the indicated antibodies. (B) HEK293 cells were transfected either with G12V mutant KRAS in the presence or absence of SM2-CA. Twelve hours post-transfection, cells were treated with cycloheximide (50 μg/ml), and cell lysates were harvested at the indicated time points and analyzed by immunoblot analysis with the antibodies mentioned. (C) Graphical representation of the quantification of KRAS protein levels shown in B to determine protein half-life. Relative KRAS levels were determined by densitometric scanning of the representative immunoblot considering 0 hour band intensity as 1 (arbitrary units). (D) Endogenous KRAS half-life was determined for H2347 and H441 cells on Smurf2 knockdown. Indicated cells were transfected with Smurf2 siRNA, and 48 hours post-transfection, cells were treated with cycloheximide (50 μg/ml), harvested at indicated times, and analyzed by immunoblot analysis. (E) Protein half-lives were determined as explained in C. Each value represents protein intensity average ± SD from three independent experiments and plotted on a log-linear scale.

SMURF2 destabilizes β-TrCP1 to indirectly protect mutant KRAS from degradation. (A) H441 cells were transfected with either control or β-TrCP1 (βT1) siRNA before Smurf2 (S) knockdown. Twenty-four hours post-transfection, cell lysates were subjected to immunoblot analysis. (B) H358 and H441 cells were transfected with either C or S siRNA, and cell lysates were subjected to immunoblot analysis. (C) H441 cells were transfected with either control (C) or Smurf2 (S) siRNA. Twenty-four hours post-transfection, cells were treated with 50 μg/ml cycloheximide (CHX) for the indicated times. Cell lysates were prepared and subjected to immunoblot analysis. (D) Protein half-life of β-TrCP1 was determined as explained above. (E) HEK293 cells were transfected with either wild type or catalytically inactive (CA) mutants of SMURF2 along with β-TrCP1 as indicated. Twenty-four hours post-transfection, cell lysates were prepared and subjected to immunoblot analysis. (F) H441 cells were transfected with β-TrCP1 either alone or in combination with SMURF2 (either wild type or CA mutants). Forty-eight hours post-transfection, cells were treated with MG132, and 4 hours post-treatment, cell lysates were subjected to immunoprecipitation with β-TrCP1 antibodies followed by immunoblot analysis using indicated antibodies. (G) In vitro ubiquitination assays were performed using immunoprecipitated Flag-tagged β-TrCP1 in the presence or absence of purified recombinant SMURF2. UBCH5, a known E2 for SMURF2, was used in the reaction. Immunoblot analyses were performed using indicated antibodies.