contributed equally to this work Insulin stimulates glucose uptake in muscle and adipose cells by mobilizing intracellular membrane vesicles containing GLUT4 glucose transporter proteins to the plasma membrane. Here we show in live cultured adipocytes that intracellular membranes containing GLUT4±yellow¯uorescent protein (YFP) move along tubulin±cyan¯uorescent protein-labeled microtubules in response to insulin by a mechanism that is insensitive to the phosphatidylinositol 3 (PI3)-kinase inhibitor wortmannin. Insulin increased by several fold the observed frequencies, but not velocities, of long-range movements of GLUT4±YFP on microtubules, both away from and towards the perinuclear region. Genomics screens show conventional kinesin KIF5B is highly expressed in adipocytes and this kinesin is partially co-localized with perinuclear GLUT4. Dominant-negative mutants of conventional kinesin light chain blocked outward GLUT4 vesicle movements and translocation of exofacial Myc-tagged GLUT4±green¯uorescent protein to the plasma membrane in response to insulin. These data reveal that insulin signaling targets the engagement or initiates the movement of GLUT4-containing membranes on microtubules via conventional kinesin through a PI3-kinase-independent mechanism. This insulin signaling pathway regulating KIF5B function appears to be required for GLUT4 translocation to the plasma membrane.
Cancer cells undergo epithelial-mesenchymal transition (EMT) as a program of increased invasion and metastasis during cancer progression. Here, we report that a novel regulator of EMT in cancer cells is protein kinase D1 (PKD1), which is downregulated in advanced prostate, breast, and gastric cancers. Ectopic reexpression of PKD1 in metastatic prostate cancer cells reversibly suppressed expression of mesenchyme-specific genes and increased epithelial markers such as E-cadherin, whereas small interfering RNA-mediated knockdown of PKD1 increased expression of mesenchyme markers. Further, PKD1 inhibited tumor growth and metastasis in a tumor xenograft model. PKD1 phosphorylates Ser 11 (S11) on transcription factor Snail, a master EMT regulator and repressor of E-cadherin expression, triggering nuclear export of Snail via 14-3-3σ binding. Snail S11 mutation causes acquisition of mesenchymal traits and expression of stem cell markers. Together, our results suggest that PKD1 functions as a tumor and metastasis suppressor, at least partly by regulating Snail-mediated EMT, and that loss of PKD1 may contribute to acquisition of an aggressive malignant phenotype.
B-Catenin is essential for E-cadherin-mediated cell adhesion in epithelial cells and also acts as a key cofactor for transcription activity. We previously showed that protein kinase D1 (PKD1), founding member of the PKD family of signal transduction proteins, is down-regulated in advanced prostate cancer and interacts with E-cadherin. This study provides evidence that PKD1 interacts with and phosphorylates B-catenin at Thr 112 and Thr 120 residues in vitro and in vivo; mutation of Thr 112 and Thr 120 results in increased nuclear localization of B-catenin and is associated with altered B-catenin-mediated transcription activity. It is known that mutation of Thr 120 residue abolishes binding of B-catenin to A-catenin, which links to cytoskeleton, suggesting that PKD1 phosphorylation of Thr 120 could be critical for cell-cell adhesion. Overexpression of PKD1 represses B-catenin-mediated transcriptional activity and cell proliferation. Epistatic studies suggest that PKD1 and E-cadherin are within the same signaling pathway. Understanding the molecular basis of PKD1-B-catenin interaction provides a novel strategy to target B-catenin function in cells including prostate cancer.
We and others previously demonstrated that Protein Kinase D1 (PKD1) is down regulated in several cancers including prostate, interacts with E-cadherin, a major cell adhesion epithelial protein and causes increased cell aggregation and decreased motility of prostate cancer cells. In this study, we demonstrate that PKD1 complexes with β3-integrin resulting in activation of Mek-Erk pathway, which causes increased production of MMP-2 and -9, that is associated with shedding of soluble 80 kDa E-cadherin extracellular domain. Interestingly, decreased cell proliferation following PKD1 transfection was rescued by MMP-2 and MMP-9 inhibitors and augmented by recombinant MMP-2 and MMP-9 proteins, suggesting an anti-proliferative role for MMPs in prostate cancer. Translational studies by in silico analysis of publicly available DNA microarray data sets demonstrate a significant direct correlation between PKD1 and MMP-2 expression in human prostate tissues. The study demonstrates a novel mechanism for anti-proliferative effects of PKD1, a protein of emerging translational interest in several human cancers, through increased production of MMP-2 and -9 in cancer cells.
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