NE-AUA 2006 Annual Meeting, September 28 - 30, 2006, The Westin Hotel & Rhode Island Convention Center Providence, Rhode Island
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Protein Kinase D1 is Associated with Membrane Trafficking of β-catenin
Meena Jaggi, PhD1, K.C. Balaji, MD2.
1University of South Dakota, Sioux Falls, SD, USA, 2University of Massachusetts Memorial Medical Center, Worcester, MA,

Introduction and objectives: We have previously published that Protein Kinase D1 (PKD1), a serine/threonine kinase that is down regulated in advanced prostate cancer (PC) (Jaggi et al., BBRC, 2003, 307, p25,) and demonstrated that PKD1 interacts with the E-cadherin-catenin complex of proteins, which play a major role in cell adhesion and proliferation (Jaggi et al., Cancer Research 2005, 65: (2), 483-492). Our more recent study has identified a novel and direct interaction between kinase domain of PKD1 and β-catenin. In this study, we establish the cellular functional significance of PKD1-β-catenin interaction in PC.
Methods: C4-2 cells were transfected with green fluorescent protein (GFP) fused PKD1 to study the effect of Bryostatin-1, a natural macrocyclic lactone that is a PKD modulator, on β-catenin subcellular localization. C4-2 -GFP-PKD1 cells were grown for 24 h and treated with 10 nM Bryostatin-1 for various time points, and cell lysates were made. Phosphorylation of PKD1 was determined by Western blot analysis using phosphorylation site-specific antibodies. Nuclear cellular extracts were made in HEPES buffer (hypertonic and hypotonic) and membrane and cytoplasmic extracts were made in Tris buffer. Proteins were resolved by SDS-PAGE, followed by immunoblotting. Further colocalization experiments were carried out using Golgi and vesicular transport specific antibodies (Golgi Sampler Kit, BD Transduction laboratories, Palo Alto, CA) to study the influence of PKD1 on β-catenin Golgi to membrane trafficking by confocal Laser Scanning Microscopy (LSM).
Results: Bryostatin-1 treatment (30 min of 10 nM) induces PKD1 activation, which is mediated through phosphorylation of two activation loop sites, Ser744 and Ser748 transphosphorylation and increased autophosphorylation of the carboxy terminal Ser916 in C4-2 cells transfected with GFP-PKD1 expression vector. Activation by Bryostatin-1 is associated with translocation of PKD1 to cell membrane in C4-2-GFP-PKD1 cells. PKD1 and β-catenin colocalized at trans Golgi network (p230 trans Golgi) and transport vesicles to plasma membrane suggesting that PKD1 is associated membrane trafficking of β-catenin(Syntaxin 11). In addition to translocation of β-catenin to membrane, activation of PKD1 by Bryostatin-1 caused decrease in nuclear β-catenin, suggesting a functional role for PKD1-β-catenin interaction in PC.
Conclusion: Our study has identified a novel and direct interaction between kinase domain of PKD1 and β-catenin resulting in membrane trafficking of β-catenin. This interaction may be functionally important in PC because PKD1 activation influences membrane trafficking and nuclear localization of β-catenin. The down regulation of PKD1 in advanced PC may contribute to PC progression by altering β-catenin function.


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