Journal: Science (New York, N.Y.)
Article Title: Reactivation of PTEN tumor suppressor for cancer treatment through inhibition of a MYC-WWP1 inhibitory pathway
Figure Lengend Snippet: Identification and characterization of the WWP1 E3 ligase for PTEN K27-linked polyubiquitination. ( A ) Lysates from DU145 cells transfected with hemagglutinin (HA)–PTEN were immunoprecipitated (IP) with an anti-PTEN antibody followed by mass spectrometric peptide sequencing. WWP1 and PTEN were identified. IgG, immunoglobulin G. ( B ) WWP1 interacts with PTEN endogenously. DU145 cells were immunoprecipitated with either anti-WWP1 or anti-PTEN antibody and then analyzed by Western blot (WB). Input is 5% of the total lysates used in IP. ( C ) Analysis of endogenous PTEN ubiquitination (Ub) in Wwp1 −/− MEFs with stable reconstitution of either WWP1 WT or its catalytically inactive mutant (C890A, abbreviated CA). ( D and E ) Effects of the indicated ubiquitin KR (Lys to Arg) (D) or K-only (E) ubiquitin mutants on WWP1-mediated PTEN polyubiquitination. HEK293T cells were transfected with the indicated constructs, and PTEN ubiquitination was analyzed. The ubiquitinated proteins were pulled down under denaturing conditions by nickel–nitrilotriacetic acid (Ni-NTA) agarose and analyzed by Western blot. ( F ) Analysis of PTEN K27-linked polyubiquitination in PC3 cells expressing the indicated NEDD4 family ubiquitin ligases as in (D). EV, cells transfected with empty vector plasmids. ( G ) In vitro ubiquitination of PTEN by WWP1 E3 ligase. Flag-PTEN purified from HEK293 cells was subject to in vitro ubiquitination reaction in the presence of E1, E2, E3, and ubiquitin or various ubiquitin mutants and then examined by Western blot with anti-PTEN antibody. The input control of WWP1 was determined by Western blotting and is shown at the bottom. ( H ) Analysis of endogenous PTEN ubiquitination by small interfering RNA (siRNA)–mediated WWP1 knock down in U2OS ubiquitin replacement cells treated with 1 μg/ml doxycycline for 48 hours. shUb, shRNAs targeting ubiquitin. ( I ) Effects of the indicated ubiquitin KR mutants on WWP1-mediated PTEN polyubiquitination. PC3 cells were transfected with indicated constructs, and PTEN ubiquitination was analyzed as in (D). In (D), (F), (H), and (I), actin is used as a loading control.
Article Snippet: The two individual siRNA duplexes targeted to WWP1 and control nontarget siRNA were purchased from Sigma Aldrich, whereas siRNA SMARTpool targeted to MYC was purchased from Dharmacon.
Techniques: Transfection, Immunoprecipitation, Sequencing, Western Blot, Mutagenesis, Construct, Expressing, Plasmid Preparation, In Vitro, Purification, Small Interfering RNA