PTEN Acetylation Modulates Its Interaction with PDZ Domain Tsuneo Ikenoue, 1 Ken Inoki, 1 Bin Zhao, 2,3 and Kun-Liang Guan 1,2,3 1 Life Sciences Institute and 2 Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan and 3 Department of Pharmacology and the Moores Cancer Center, University of California at San Diego, La Jolla, California Abstract The PTEN tumor suppressor gene is frequently inactivated in human cancer. As a major tumor suppressor, PTEN function must be tightly regulated. Both phosphorylation and mem- brane association have been reported to regulate PTEN activity. In addition, the COOH terminus of PTEN has a typical PDZ domain-binding motif that interacts with several PDZ domain-containing proteins. In this report, we show that PTEN is acetylated on Lys 402 , which is in the COOH- terminal PDZ domain-binding motif. We show that CBP plays a major role in PTEN acetylation, whereas the SIRT1 deacetylase is mainly responsible for PTEN deacetylation. Interestingly, Lys 402 acetylation modulates PTEN interaction with PDZ domain-containing proteins, indicating a potential role of acetylation in regulating PTEN function. [Cancer Res 2008;68(17):6908–12] Introduction PTEN, phosphatase and tensin homologue deleted in chromo- some 10, is the second most frequently mutated tumor suppressor gene in f50% of human cancer. The mechanism of PTEN in tumor suppressor function has been clearly defined by its biochemical activity as a lipid phosphatase (1–3). PTEN specifically dephosphorylates the three position of phosphatidy- linositols (PI3P, PI3,4P2, and PI3,4,5P3), which are the product of phosphatidylinositol 3-kinase (PI3K). Therefore, PTEN reverses the biological functions of PI3K, which is activated by numerous growth-stimulating signals, such as mitogenic growth factors. Activation of PI3K plays a critical role in the mitogenic and antiapoptotic effects of growth factors. Although extensive studies have been conducted to show the tumor suppressor function of PTEN, much less is known about PTEN regulation. It is clear that membrane localization of PTEN is important for its biological function as the PTEN substrates are constituents of membrane. The C2 domain of PTEN plays an important role in membrane localization (4–6). Furthermore, the COOH terminus of PTEN contains a typical PSD-95/Dlg/ZO-1 (PDZ) binding motif. Indeed, PTEN has been reported to associate with several PDZ domain-containing proteins and these interactions may play a role in regulating the biological function of PTEN (7–10). However, it is not clear whether and how the association between PTEN and its PDZ domain-containing receptors are regulated. Protein acetylation is an important posttranslational regulatory mechanism (11). Most of the studies of protein acetylation focus on histone and nuclear transcription regulators (12, 13). Much less is known about lysine acetylation for cytoplasmic proteins. Interest- ingly, it has been reported that PTEN is acetylated on Lys 125 and Lys 128 by PCAF, a histone acetyltransferase (14). In this report, we observed that PTEN is acetylated on Lys 402 , which is localized in the PDZ domain-binding motif TKV. We identified CBP and SIRT1 as the major acetyltransferase and deacetylase in controlling PTEN acetylation. Our data indicate that acetylation may regulate the interaction between PTEN and PDZ domain-containing proteins. Materials and Methods Antibodies and reagents. PTEN and acetyl-lysine antibodies were purchased from Cell Signaling. HA and Myc antibodies were from Covance. Nicotinamide,trichostatinA,andothercommonchemicalswerefromSigma. Plasmids. Human PTEN cDNA was provided by Dr. Jack E. Dixon (University of California at San Diego, La Jolla, CA). PTEN was subcloned into pRK5-Myc and pRK7-HA vectors to create Myc-PTEN and HA-PTEN expression constructs. Human PCAF was cloned into pCDNA3-FLAG vector to create FLAG-PCAF. HA-SIRT1, SIRT2, FLAG-CBP, and p300 plasmids are provided by Dr. Roland P. Kwok (University of Michigan, Ann Arbor, MI). Human discs large DLG (hDLG) and mouse membrane-associated guanylate kinase inverted-2 (MAGI-2) were provided by Dr. Ben Magolis (University of Michigan) and Dr. Hiromu Sugino (University of Tokushima, Tokushima, Japan), respectively. To create bacterial expression constructs, the fragments containing the second PDZ domain (PDZ2) of hDLG and MAGI-2 were subcloned into pGEX-KG vector. Small interfering RNA. siGENOME SMARTpool small interfering RNA (siRNA) targeting either SIRT1, SIRT2, CBP, p300, or PCAF was purchased from Dharmacon. Efficiency of siRNA on each target was examined by quantitative reverse transcription-PCR (RT-PCR; Figs. 1D and 2C). RNA isolation and real-time PCR. Total RNA was isolated from cultured cells using Trizol reagent (Invitrogen) and subjected to real-time RT-PCR in the presence of Cybergreen (Applied Biosystems). Relative abundance of mRNA was normalized to h-actin mRNA. Cell culture and transfection. COS7 cells were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin. Transfection was performed using Lipofectamine (Invitrogen). Immunoprecipitation. Cells were harvested in ice-cold mild lysis buffer [10 mmol/L Tris-HCl (pH 7.5), 100 mmol/L NaCl, 1% NP40, 50 mmol/L NaF, 2 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 10 Ag/mL leupeptin, 10 Ag/mL aprotinin]. The lysates were incubated with HA or Myc antibody for 1 h at 4jC followed by addition of protein G-Sepharose beads and further incubation for overnight. The immunoprecipitates were washed four times with the lysis buffer. The samples were then subjected to SDS- PAGE and analyzed by immunoblotting. Glutathione S -transferase pull-down. The glutathione S -transferase (GST) fusion proteins containing the PDZ domain of hDLG or MAGI-2 were expressed in Escherichia coli and purified. COS7 cells transfected with HA-PTEN wild-type (WT) or PDZ-binding motif mutants were lysed with mild lysis buffer, and the lysates were incubated with the GST fusion PDZ domain for 1 h at 4jC followed by addition of glutathione-Sepharose beads and further incubation for 2 h. The beads were then washed five times with mild lysis buffer. HA-PTEN proteins in the lysates unbound to the beads were immunoprecipitated with HA antibody. Quantification of immuno- blots was done by densitometric analysis using NIH ImageJ software. PTEN phosphatase assay. HEK293 cells were transfected with HA- PTEN WT or PDZ-binding motif mutants or phosphatase inactive mutant (C124S). HA-PTEN was immunoprecipitated and phosphatase activity was Requests for reprints: Kun-Liang Guan, Department of Pharmacology and the Moores Cancer Center, University of California at San Diego, La Jolla, CA 92093-0815. Phone: 858-822-7945; Fax: 858-534-7638; E-mail: kuguan@ucsd.edu. I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-08-1107 Cancer Res 2008; 68: (17). September 1, 2008 6908 www.aacrjournals.org Priority Report Research. on December 2, 2015. © 2008 American Association for Cancer cancerres.aacrjournals.org Downloaded from