Protein Kinase D Potentiates DNA Synthesis and Cell Proliferation Induced by Bombesin, Vasopressin, or Phorbol Esters in Swiss 3T3 Cells

Department of Medicine, School of Medicine and Molecular Biology Institute, UCLA, Los Angeles, CA 90095, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2001; 276(43):40298-305. DOI: 10.1074/jbc.M106512200
Source: PubMed


We examined whether protein kinase D (PKD) overexpression in Swiss 3T3 cells potentiates the proliferative response to either
the G protein-coupled receptor agonists bombesin and vasopressin or the biologically active phorbol ester phorbol 12,13-dibutyrate
(PDBu). In order to generate Swiss 3T3 cells stably overexpressing PKD, cultures of these cells were infected with retrovirus
encoding murine PKD and green fluorescent protein (GFP) expressed as two separate proteins translated from the same mRNA.
GFP was used as a marker for selection of PKD-positive cells. PKD overexpressed in Swiss 3T3 cells was dramatically activated
by cell treatment with bombesin or PDBu as judged by in vitrokinase autophosphorylation assays and exogenous substrate phosphorylation. Concomitantly, these stimuli induced PKD phosphorylation
at Ser744, Ser748, and Ser916. PKD activation and phosphorylation were prevented by exposure of the cells to protein kinase C-specific inhibitors. Addition
of bombesin, vasopressin, or PDBu to cultures of Swiss 3T3 cells overexpressing PKD induced a striking increase in DNA synthesis
and cell number compared with cultures of Swiss 3T3-GFP cells. In contrast, stimulation of DNA synthesis in response to epidermal
growth factor, which acts via protein kinase C/PKD-independent pathways, was not enhanced. Our results demonstrate that overexpression
of PKD selectively potentiates mitogenesis induced by bombesin, vasopressin, or PDBu in Swiss 3T3 cells.

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    • "Rapid PKC-dependent PKD1 activation is followed by a late, PKC-independent phase of activation induced by Gq-coupled receptor agonists [36]–[38]. Accumulating evidence demonstrate that the PKD family plays an important role in several cellular processes and activities [23], including stimulation of DNA synthesis and proliferation [35]–[37], [39]–[41]. Indeed, PKD1 activation plays a critical role in mediating GPCR-induced migration and proliferation in non-transformed intestinal epithelial IEC-6 and IEC-18 cells [37], [42]. In these cells, we also demonstrated rapid and transient Akt activation via GPCR-induced EGFR transactivation [6]. "
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    ABSTRACT: We examined whether protein kinase D1 (PKD1) mediates negative feeback of PI3K/Akt signaling in intestinal epithelial cells stimulated with G protein-coupled receptor (GPCR) agonists. Exposure of intestinal epithelial IEC-18 cells to increasing concentrations of the PKD family inhibitor kb NB 142-70, at concentrations that inhibited PKD1 activation, strikingly potentiated Akt phosphorylation at Thr(308) and Ser(473) in response to the mitogenic GPCR agonist angiotensin II (ANG II). Enhancement of Akt activation by kb NB 142-70 was also evident in cells with other GPCR agonists, including vasopressin and lysophosphatidic acid. Cell treatment rovincial Hospital Affiliated to Shandong University, Jinan, China with the structurally unrelated PKD family inhibitor CRT0066101 increased Akt phosphorylation as potently as kb NB 142-70. Knockdown of PKD1 with two different siRNAs strikingly enhanced Akt phosphorylation in response to ANG II stimulation in IEC-18 cells. To determine whether treatment with kb NB 142-70 enhances accumulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in the plasma membrane, we monitored the redistribution of Akt-pleckstrin homology domain-green fluorescent protein (Akt-PH-GFP) in single IEC-18 cells. Exposure to kb NB 142-70 strikingly increased membrane accumulation of Akt-PH-GFP in response to ANG II. The translocation of the PIP3 sensor to the plasma membrane and the phosphorylation of Akt was completed prevented by prior exposure to the class I p110α specific inhibitor A66. ANG II markedly increased the phosphorylation of p85α detected by a PKD motif-specific antibody and enhanced the association of p85α with PTEN. Transgenic mice overexpressing PKD1 showed a reduced phosphorylation of Akt at Ser(473) in intestinal epithelial cells compared to wild type littermates. Collectively these results indicate that PKD1 activation mediates feedback inhibition of PI3K/Akt signaling in intestinal epithelial cells in vitro and in vivo.
    Full-text · Article · Sep 2013 · PLoS ONE
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    • "However, PKD1 possesses distinct substrate specificity [48] [49] and has therefore recently been classified as a novel subgroup of the calcium/calmodulin-dependent kinase (CAMK) family [39]. PKD1, which is expressed in most tissues [20] [48], has actually been reported to play a role in cell proliferation [37] [53] [58] and survival [43]. However, little is known about the downstream effectors regulated by PKD1. "
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    Full-text · Article · Feb 2012 · Biochimica et Biophysica Acta
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    • "Our results are consistent with a previous study suggesting that PKD1 tyr 469 phosphorylation does not occur in H2O2-treated Swiss 3T3 cells [48]. Similarly, it was shown that Vasopressin a GPCR agonist can activates PKD1 in Swiss 3T3 cells [30]. Since H2O2 did not phosphorylate tyr 469, we used desmopressin, a synthetic analogue of vasopressin to induce tyr 469 phosphorylation. "
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    ABSTRACT: Oxidative stress is a key pathophysiological mechanism contributing to degenerative processes in many neurodegenerative diseases and therefore, unraveling molecular mechanisms underlying various stages of oxidative neuronal damage is critical to better understanding the diseases and developing new treatment modalities. We previously showed that protein kinase C delta (PKCδ) proteolytic activation during the late stages of oxidative stress is a key proapoptotic signaling mechanism that contributes to oxidative damage in Parkinson's disease (PD) models. The time course studies revealed that PKCδ activation precedes apoptotic cell death and that cells resisted early insults of oxidative damage, suggesting that some intrinsic compensatory response protects neurons from early oxidative insult. Therefore, the purpose of the present study was to characterize protective signaling pathways in dopaminergic neurons during early stages of oxidative stress. Herein, we identify that protein kinase D1 (PKD1) functions as a key anti-apoptotic kinase to protect neuronal cells against early stages of oxidative stress. Exposure of dopaminergic neuronal cells to H2O2 or 6-OHDA induced PKD1 activation loop (PKD1S744/748) phosphorylation long before induction of neuronal cell death. Blockade of PKCδ cleavage, PKCδ knockdown or overexpression of a cleavage-resistant PKCδ mutant effectively attenuated PKD1 activation, indicating that PKCδ proteolytic activation regulates PKD1 phosphorylation. Furthermore, the PKCδ catalytic fragment, but not the regulatory fragment, increased PKD1 activation, confirming PKCδ activity modulates PKD1 activation. We also identified that phosphorylation of S916 at the C-terminal is a preceding event required for PKD1 activation loop phosphorylation. Importantly, negative modulation of PKD1 by the RNAi knockdown or overexpression of PKD1S916A phospho-defective mutants augmented oxidative stress-induced apoptosis, while positive modulation of PKD1 by the overexpression of full length PKD1 or constitutively active PKD1 plasmids attenuated oxidative stress-induced apoptosis, suggesting an anti-apoptotic role for PKD1 during oxidative neuronal injury. Collectively, our results demonstrate that PKCδ-dependent activation of PKD1 represents a novel intrinsic protective response in counteracting early stage oxidative damage in neuronal cells. Our results suggest that positive modulation of the PKD1-mediated compensatory protective mechanism against oxidative damage in dopaminergic neurons may provide novel neuroprotective strategies for treatment of PD.
    Full-text · Article · Jun 2011 · Molecular Neurodegeneration
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