Protein kinase D (PKD) activation in intact cells through a protein kinase C-dependent signal transduction pathway.

Abstract

Protein kinase D (PKD) is a serine/threonine protein kinase that is directly stimulated in vitro by phorbol esters and diacylglycerol in the presence of phospholipids. Here, we examine the regulation of PKD in living cells. Our results demonstrate that tumour‐promoting phorbol esters, membrane‐permeant diacylglycerol and serum growth factors rapidly induced PKD activation in immortalized cell lines (e.g. Swiss 3T3 and Rat‐1 cells), in secondary cultures of mouse embryo fibroblasts and in COS‐7 cells transiently transfected with a PKD expression construct. PKD activation was maintained during cell disruption and immunopurification and was associated with an electrophoretic mobility shift and enhanced 32P incorporation into the enzyme, but was reversed by treatment with alkaline phosphatase. PKD was activated, deactivated and reactivated in response to consecutive cycles of addition and removal of PDB. PKD activation was completely abrogated by exposure of the cells to the protein kinase C inhibitors GF I and Ro 31–8220. In contrast, these compounds did not inhibit PKD activity when added directly in vitro. Co‐transfection of PKD with constitutively activated mutants of PKCs showed that PKCepsilon and eta but not PKCzeta strongly induced PKD activation in COS‐7 cells. Thus, our results indicate that PKD is activated in living cells through a PKC‐dependent signal transduction pathway.

The zinc-finger transcription factor, early growth response 3, mediates VEGF-induced angiogenesis

“…*Po0.05, **Po0.01 for inhibitor vs VEGF alone. (Zugaza et al, 1996). VEGF induced a striking increase in PKD phosphorylation at serines 744 and 748, essential for PKC-mediated activation of PKD.…”

Protein Kinase D Controls the Integrity of Golgi Apparatus and the Maintenance of Dendritic Arborization in Hippocampal Neurons

“…Phorbol ester treatment is known to cause plasma membrane translocation and activation of PKD (Zugaza et al, 1996;Matthews et al, 1999;Rey et al, 2004). In live cell imaging of hippocampal neurons, 1 M PDBu treatment led to a rapid recruitment of wtPKD1-EGFP to the plasma membrane, followed by the intracellular accumulation of the fluorescently tagged protein ( Figure 1B).…”

“…Besides a rather homogenous cytoplasmic distribution, wtPKD1-EGFP was significantly enriched at the Golgi apparatus (Table 1). On closer examination, wtPKD1-EGFP was localized side by side with GM130 but showed partially overlapping localization with VAMP4-stained structures ( Figure 1A, enlargements).Phorbol ester treatment is known to cause plasma membrane translocation and activation of PKD (Zugaza et al, 1996;Matthews et al, 1999;Rey et al, 2004). In live cell imaging of hippocampal neurons, 1 M PDBu treatment led to a rapid recruitment of wtPKD1-EGFP to the plasma membrane, followed by the intracellular accumulation of the fluorescently tagged protein ( Figure 1B).…”

Identification of a novel phosphorylation site in c-jun directly targeted in vitro by protein kinase D

“…Wild-type pcDNA3-PKD [14] and kinase-deficient pcDNA3-PKDK618N [15] were described previously. 3XHA-JNK1α1 and pSRα3-3XHA-JNK2α2 were gifts from R. Chiu, UCLA and M. Karin, UCSD, respectively.…”

“…After 72 h, cells expressing PKD forms were left untreated or treated with PDB to induce PKD activation [15], and lysed in buffer (1% Triton X-100, 2 mM EGTA, 2 mM EDTA in 50 mM Tris-HCl, pH 7.4, with 2 mM dithiothreitol, 1 μg/ml aprotinin, 10 μg/ ml leupeptin and 1 mM AEBSF). To induce JNK activation [1], cells were stimulated with UV light (5000 μJ/m2) for 3.5 min and incubated at 37 °C for 15 min, washed with ice-cold PBS, and extracted in buffer comprising 0.1% Triton X-100 and (in mM) 25 Hepes, 300 NaCl, 1.5 MgCl 2 , 0.2 EDTA, 0.5 DTT, 20 β-glycerophosphate, 0.1 Na 2 VO 4 .…”