Abstract
Rapid protein kinase D (PKD) activation and phosphorylation via protein kinase C (PKC) have been extensively documented in many cell types cells stimulated by multiple stimuli. In contrast, little is known about the role and mechanism(s) of a recently identified sustained phase of PKD activation in response to G protein-coupled receptor agonists. To elucidate the role of biphasic PKD activation, we used Swiss 3T3 cells because PKD expression in these cells potently enhanced duration of ERK activation and DNA synthesis in response to G(q)-coupled receptor agonists. Cell treatment with the preferential PKC inhibitors GF109203X or Gö6983 profoundly inhibited PKD activation induced by bombesin stimulation for <15 min but did not prevent PKD catalytic activation induced by bombesin stimulation for longer times (>60 min). The existence of sequential PKC-dependent and PKC-independent PKD activation was demonstrated in 3T3 cells stimulated with various concentrations of bombesin (0.3-10 nm) or with vasopressin, a different G(q)-coupled receptor agonist. To gain insight into the mechanisms involved, we determined the phosphorylation state of the activation loop residues Ser(744) and Ser(748). Transphosphorylation targeted Ser(744), whereas autophosphorylation was the predominant mechanism for Ser(748) in cells stimulated with G(q)-coupled receptor agonists. We next determined which phase of PKD activation is responsible for promoting enhanced ERK activation and DNA synthesis in response to G(q)-coupled receptor agonists. We show, for the first time, that the PKC-independent phase of PKD activation mediates prolonged ERK signaling and progression to DNA synthesis in response to bombesin or vasopressin through a pathway that requires epidermal growth factor receptor-tyrosine kinase activity. Thus, our results identify a novel mechanism of G(q)-coupled receptor-induced mitogenesis mediated by sustained PKD activation through a PKC-independent pathway.
Highlights
The understanding of the mechanisms that control cell proliferation requires the identification of the molecular pathways that govern the transition of quiescent cells into the S phase of the cell cycle
In response to cellular stimuli [1], including phorbol esters, growth factors (e.g. PDGF), and G protein-coupled receptor (GPCR) agonists (6, 8 –16) that signal through Gq, G12, Gi, and Rho [11, 15,16,17,18,19], protein kinase D (PKD) is converted into a form with high catalytic activity, as shown by in vitro kinase assays performed in the absence of lipid co-activators [5, 20]
protein kinase C (PKC)-independent PKD Activation Mediates Mitogenic Signaling pendent PKD activation was followed by a late, PKC-independent phase of catalytic activation and phosphorylation induced by stimulation of the bombesin Gq-coupled receptor ectopically expressed in COS-7 cells [22]
Summary
Stock cultures of Swiss 3T3-PKD.GFP cells, which overexpress wild type PKD, Swiss 3T3-PKDK618N.GFP cells, that overexpress a kinase-deficient PKD, and control Swiss 3T3GFP cells were generated as previously described [8, 14]. Cells were plated in 100-mm dishes at 6 ϫ 105 cells/dish or 35-mm dishes at 1 ϫ 105 cells/dish and grown in DMEM containing 10% fetal bovine serum for 7–9 days until they became confluent and quiescent [35]. Quiescent Swiss 3T3-GFP, Swiss 3T3-PKD.GFP, and Swiss 3T3-PKDK618N.GFP cells were lysed in 2ϫ SDSPAGE sample buffer (20 mM Tris/HCl, pH 6.8, 6% SDS, 2 mM EDTA, 4% 2-mercaptoethanol, 10% glycerol) and boiled for 10 min. After SDS-PAGE, proteins were transferred to Immobilon-P membranes. Membranes were blocked using 5% nonfat dried milk in phosphate-buffered saline, pH 7.2, and incubated for at least 2 h with the desired antibodies diluted in phosphate-buffered saline, pH 7.2, containing 3% nonfat dried milk. Autoluminograms were scanned using a GS-710 scanner (Bio-Rad), and the labeled bands were quantified using the Quantity One software program (Bio-Rad)
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