Protein Kinase D1–Mediated Phosphorylation and Subcellular Localization of B-Catenin
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Published OnlineFirst January 13, 2009; DOI: 10.1158/0008-5472.CAN-07-6270 Published Online First on January 13, 2009 as 10.1158/0008-5472.CAN-07-6270 Research Article Protein Kinase D1–Mediated Phosphorylation and Subcellular Localization of B-Catenin Cheng Du,1 Meena Jaggi,2 Chuanyou Zhang,1 and K.C. Balaji1 1Division of Urology, Department of Surgery, University of Massachusetts Medical School, Worcester, Massachusetts and 2Department of Obstetrics and Gynecology and Basic Biomedical Science, Sanford School of Medicine, The University of South Dakota, Sioux Falls, South Dakota Abstract balancing between these functions is crucial for normal develop- B ment, and the wrong choice can lead to cancer. -Catenin is essential for E-cadherin–mediated cell adhesion h in epithelial cells and also acts as a key cofactor for Molecular switches of the dual role of -catenin are tightly transcription activity. We previously showed that protein regulated. The structural and functional integrity of the cadherin- catenin complex is mainly regulated by phosphorylation. Casein kinase D1 (PKD1), founding member of the PKD family of h signal transduction proteins, is down-regulated in advanced kinase II phosphorylates -catenin (9) and E-cadherin (10) on prostate cancer and interacts with E-cadherin. This study serine and threonine residues. This phosphorylation leads to increased stabilization of the cadherin-catenin complex. Phosphor- provides evidence that PKD1 interacts with and phosphor- h ylates B-catenin at Thr112 and Thr120 residues in vitro and ylation by AKTkinase causes -catenin disassociation from cell- in vivo 112 120 cell contacts and accumulation in both the cytosol and the nucleus, ; mutation of Thr and Thr results in increased h nuclear localization of B-catenin and is associated with increases -catenin transcriptional activity, and promotes tumor altered B-catenin–mediated transcription activity. It is known cell invasion (11). On the other hand, tyrosine phosphorylation of h h a that mutation of Thr120 residue abolishes binding of B-catenin -catenin disrupts binding of the E-cadherin/ -catenin/ -catenin to A-catenin, which links to cytoskeleton, suggesting that complex (12–15). In contrast, activation of protein phosphatases PKD1 phosphorylation of Thr120 could be critical for cell-cell stabilizes the cadherin-catenin complex and results in increased adhesion. Overexpression of PKD1 represses B-catenin–medi- cadherin-mediated cell-cell adhesion (16, 17). ated transcriptional activity and cell proliferation. Epistatic Protein kinase D1 (PKD1), founding member of a new family of studies suggest that PKD1 and E-cadherin are within the same serine/threonine protein kinases, lies downstream of the signal signaling pathway. Understanding the molecular basis of transduction pathways initiated by diacylglycerol and protein kinase C. Diacylglycerol regulates the intracellular localization of PKD1–B-catenin interaction provides a novel strategy to target B-catenin function in cells including prostate cancer. PKD1 and also activates PKD1 through protein kinase C by phosphorylation. Prior studies show that PKD1 plays a role in cell [Cancer Res 2009;69(3):OF1–8] growth, gene expression, survival, motility, protein trafficking, and lymphocyte biology (18). We initially discovered that PKD1 is Introduction down-regulated in advanced prostate cancer and later described its h-Catenin was originally identified as a key effector of the interaction with E-cadherin (19, 20). In this study, we show that h Wnt/Wg signaling pathway in Drosophila (1). It has also been PKD1 directly binds to and phosphorylates -catenin and show the identified as a component of adherens junctions with the primary biological consequences of the phosphorylation. cell adhesion molecule E-cadherin (2–4). h-Catenin promotes cell adhesion by binding to the intracellular domain of the cadherin Materials and Methods adhesion receptors and linking cadherin to the actin cytoskeleton h through the adaptor protein a-catenin (reviewed in ref. 5). This Constructs. Human -catenin clone was obtained from Mammalian Gene h association with the actin-based cytoskeleton is important for the Collection. Full-length -catenin was amplified by PCR and cloned into bacterial expression vector pGEX-5X-1, yeast two-hybrid plasmid pGADT7, adhesion function of all classic cadherins, as best shown for the and mammalian expression vectors pEGFP-C1 and DsRed2-C1. Another E-cadherin-catenin complex in adherens junctions of epithelial h-catenin expression vector, h-catenin-pCS2+, was kindly provided by cells (6). Dr. R. Kemler (Department of Molecular Embryology, Max-Planck Institute h-Catenin can be found in three cell compartments: the plasma of Immunology, Freiburg, Germany; ref. 9). Site-specific mutagenesis to membrane, the cytoplasm, and the nucleus. Nuclear h-catenin acts change Thr102 to isoleucine (T102I), Thr112 to arginine (T112R), and Thr120 to as cofactor for a wide variety of transcription factors, including isoleucine (T120I) was done with a PCR-based method. Primers for T-cell factor (TCF), androgen receptor (7), and nuclear factor nB mutagenesis: forward (T102I) 5¶-GCTGCTATGTTCCCTGAGATCTTAGAT- (8). Soluble cytoplasm pool of h-catenin is highly unstable and is GAGGGCATGCAG; (T112R) 5¶-GCATGCAGATCCCATCTAGACAGTTT- ¶ stabilized by Wnt signal resulting in nucleus localization. The GATGCTGCTCATC; and (T120I) 5 -TTGATGCTGCTCATCCCATTAAT- dynamic balance of h-catenin functions in both cell adhesion and GTCCAGCGTTTGGCTG, where the underlined sequences contain mutated sites and new restriction sites to facilitate clone selection. transcription has recently won increasing interest. Properly A COOH-terminal truncated h-catenin in pEGFP-C1 (h-catN/pEGFP) was made by restriction digestion of correspondent full-length vector with Plm Sma Note: C. Du and M. Jaggi contributed equally to this work. deletion of I and I fragments. The construct includes the first 150 Requests for reprints: Cheng Du or K.C. Balaji, Division of Urology, Department of amino acid residues of h-catenin. Expression plasmid for full-length Surgery, University of Massachusetts Medical School, 55 Lake Avenue, North E-cadherin expression vector was constructed by cloning PCR product into Worcester, MA 01655. Phone: 508-856-2576; Fax: 508-856-3137; [email protected] pEGFP-N1 vector. shRNA vectors targeting PKD1 (siPKD1) and E-cadherin or [email protected]. I2009 American Association for Cancer Research. (siEcad) were described in ref. 21. All constructs were confirmed by doi:10.1158/0008-5472.CAN-07-6270 sequencing. www.aacrjournals.org OF1 Cancer Res 2009; 69: (3).February 1, 2009 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst January 13, 2009; DOI: 10.1158/0008-5472.CAN-07-6270 Cancer Research Yeast two-hybrid assay. The C1a, C1b, pleckstrin homology (PH), and Because E-cadherin forms a protein complex with h-catenin, we catalytic domains of PKD1 were cloned in pGBKT7 vector as previously tested if PKD1 affects h-catenin transcription activity. Expression described (22). Yeast strain PJ69-4A was used to detect protein interaction of h-catenin was able to increase TCF transcription activity in according to standard protocol (23). Topflash assay in NIH 3T3 cells (Fig. 1A). Ectopic expression of In vitro phosphorylation assay. Wild-type and mutant h-catenin E-cadherin or PKD1 individually inhibits h-catenin/TCF transcrip- proteins were expressed and purified from E. coli. Preparation of active and inactive PKD1 from 293Tcells transfected with PKD1 expression vectors tion activity. Expression of a kinase-dead PKD1 (PKD1-KD) has less pEGFP-PKD1 and pEGFP-PKD1-KD, respectively (20), by immunoprecipi- inhibitory effects than wild-type PKD1. Conversely, expression of tation was described (24). Briefly, transfected cells were treated with shRNAs against PKD1 (siPKD1) or E-cadherin (siEcad) increases 0.5 Amol/L phorbol 12,13-dibutyrate for 30 min before being lysed. Cells h-catenin/TCF transcription activity. Similar results were obtained were washed twice in TBS [25 mmol/L Tris (pH 7.5), 0.1 mol/L NaCl], from the colon cancer SW480 cell line, which is negative for followed by lysis in TBS, 1% Triton X-100, and protease inhibitor cocktail adenomatous polyposis coli and with high levels of h-catenin, and (Sigma Chemicals). Immunoprecipitation used anti-PKD1 antibody (C20, the prostate cancer C4-2 cell line (data not shown). The data Santa Cruz Biotechnology) and final beads were resuspended in TBS plus strongly suggest that both PKD1 and E-cadherin regulate h- In vitro A A 10% glycerol. kinase assay was carried with 5 L PKD1 kinase, 30 L catenin/TCF signaling negatively. purified protein, 5 AL kinase buffer [250 mmol/L Tris (pH 7.5), 100 mmol/L h A 32 A A The similar phenotypes of PKD1 and E-cadherin on -catenin/ MgCl2, 1 mmol/L ATP], and 1 Lof[ P]ATP (3,000 Ci/mmol) in a 50- L volume at 37jC for 1 h. TCF function led us to study their epistatic relationship. We asked Transient transfection and luciferase assay. Transfection was done by if knockdown of PKD1 could overcome the E-cadherin repression using the Lipofectamine 2000 method (Invitrogen). Luciferase dual-reporter on Topflash activity or vice versa. The siPKD1 was cotransfected assays with Super8xTopflash (25) and pRL-TK (Promega) and dual-reporter with E-cadherin expression vector or siEcad with PKD1 expression luminofluorescence assay (Promega) were done 48 h after transfection vector into mouse 3T3 cells. Knockdown of PKD1 can overcome according to the manufacturer’s recommendation. the inhibition caused by overexpression of E-cadherin and vice Immunoprecipitation and Western blots. LNCaP cells were cultured versa (Fig. 1A), suggesting that PKD1 and E-cadherin regulate in RPMI 1640 plus 10% fetal bovine serum. The cells were lysed in TBST h-catenin/TCF activity via the same pathway. (50 mmol/L Tris, pH 7.2, 100 mmol/L NaCl, 1% Triton X-100) plus protease PKD1 colocalizes and associates with B-catenin in prostate inhibitor cocktail (Sigma Chemicals). The lysate was used for immunopre- A cancer cells and human tissue.