NE-AUA 2006 Annual Meeting, September 28 - 30, 2006, The Westin Hotel & Rhode Island Convention Center Providence, Rhode Island
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Kinase Domain of Protein Kinase D1 Interacts Directly with β-catenin
Cheng Du, PhD1, Meena Jaggi, PhD2, K.C. Balaji, MD1.
1University of Massachusetts Memorial Medical Center, Worcester, MA, USA, 2Universtiy of South Dakota, Sioux Falls, SD,

Introduction: We have previously published that Protein Kinase D1 (PKD1), a serine/threonine kinase, which is down regulated in advanced prostate cancer (PC) (Jaggi et al., BBRC2003, 307, p25,) and that PKD1 interacts with the E-cadherin-catenin complex of proteins, which are known to play a major role in cell adhesion and proliferation. The PKD1-E-cadherin interaction results in E-cadherin phosphorylation by PKD1, increases cellular aggregation and decreases motility in prostate cancer (Jaggi et al., Cancer Research 2005, 65: (2), 483-492). In this study we specifically studied PKD1-β-catenin interaction in PC.
Methods. PKD1-β-catenin interaction and co localization in prostate cancer cell line and human tissue was determined by confocal Laser Scanning Microscopy (LSM) and/or immunoprecipitation (IP) assays (β-catenin antibody 5H10, obtained from Drs K. R. Johnson and M. J. Wheelock’s lab and PKD1 antibody C-20, from Santa Cruz Biotech., CA.) GST fusion protein pull-down assays and directed yeast two-hybrid analysis were used to demonstrate a direct interaction of β-catenin and PKD1. Immunostaining was done on paraffined prostate tissue sections using FITC-conjugated anti-mouse antibodies for β-catenin and rhodamine-conjugated anti-rabbit for PKD1.
Results Our study demonstrates that PKD1 co-localizes with β-catenin at cell junctions in LNCaP and IPs with PKD1 from lysates of LNCaP cells and in all five human prostate samples studied. IP experiments performed on E-cadherin negative prostate cancer cell lines (JCA and TSUPR) confirmed that β-catenin-PKD1 interaction occurred without E-cadherin and N-cadherin being present in the IP, suggesting a cadherin independent interaction. The GST pull down assay demonstrated direct interaction of PKD1-GST -fusion protein to β-catenin from SW 480 cell lysates. A yeast two-hybrid test indicated direct interaction between β-catenin and the kinase domain of PKD1.Among four domains of PKD1 (the C1, C2, PH and kinase domain) the kinase domain was found interacting with full-length β-catenin, other three domains,(the C1, C2 and PH), did not interact with β-catenin. All five human prostate samples demonstrated colocalization of PKD1 and β-catenin in glandular epithelium.
Conclusion: Our study has identified a novel and direct interaction between kinase domain of PKD1 and β-catenin. Because PKD1 and β-catenin co-localizes in human prostate cancer epithelial cells, it is highly probable that the PKD1/β-catenin interaction may also occur in human prostate and have functional consequences. Defining the specific PKD1 domains that interact directly with β-catenin will establish the scientific basis for therapeutic targeting and additional focused mechanistic studies in the future.


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