Diacylglycerol kinase is phosphorylated in vivo upon stimulation of the epidermal growth factor receptor and serine/threonine kinases, including protein kinase C-ε

Abstract

In signal transduction, diacylglycerol (DG) kinase attenuates levels of the second messenger DG by converting it to phosphatidic acid. A previously cloned full-length human 86 kDa DG kinase cDNA was expressed as fusion protein in Escherichia coli, to aid in the generation of DG-kinase-specific monoclonal antibodies suitable for immunoprecipitation experiments. To investigate whether phosphorylation of DG kinase is a possible mechanism for its regulation, COS-7 cells were transiently transfected with the DG kinase cDNA and phosphorylation of the expressed DG kinase was induced by various stimuli. Activation of both cyclic AMP-dependent protein kinase and protein kinase C (PKC) resulted in phosphorylation of DG kinase on serine residues in vivo, and both kinases induced this phosphorylation within the same tryptic phosphopeptide, suggesting that they may exert similar control over DG kinase. No phosphorylation was observed upon ionomycin treatment, intended to activate Ca2+/calmodulin-dependent kinases. Co-transfections of DG kinase with either PKC-alpha or PKC-epsilon cDNA revealed that both protein kinases, when stimulated, are able to phosphorylate DG kinase. For PKC-epsilon, DG kinase is the first in vivo substrate identified. Stimulation with epidermal growth factor (EGF) of COS-7 cells transfected with both DG kinase and EGF-receptor cDNA results mainly in phosphorylation of DG kinase on tyrosine. Since the EGF receptor has an intrinsic tyrosine kinase activity, this finding implies that DG kinase may be a direct substrate for the activated EGF receptor.

MKP5, a new member of the MAP kinase phosphatase family, which selectively dephosphorylates stress-activated kinases

“…MKP5 was subcloned into pMT-SM III, a modi®ed version of the eukaryotic expression vector pMT-SM (Muda et al, 1996a;Schaap et al, 1993), in which the 9E10 Myc epitope coding sequence is incorporated, in-frame, into the multiple cloning site. Primers TTAGGTACCTGT-CTTGAGTTCTT-CTTGAATTGC (nucleotides 74 ± 106) and CCATCTAGA-CCACGCCCATCAGC (nucleotides 1579 ± 1545) were used to amplify MKP5 ORF from human breast cDNA (the underlined nucleotides correspond to the engineered Asp718 and XbaI sites).…”

Rapid activation of sodium–proton exchange and extracellular signal-regulated protein kinase in fibroblasts by G protein-coupled 5-HT1A receptor involves distinct signalling cascades

“…Dominant negative Grb2 (∆NGrb2), in which the N-terminal SH3 domain is deleted, was from A. M. Pendergast (Duke University) [35]. Dominant negative p74 Raf-" , (N∆Raf) which only contains the Nterminal regulatory domain of Raf-1 (residues 1-258) [36], inactive p110 (p110∆kin) with a mutated inactive ATP site [37], and constitutively activated p110 (p110*) in which the p85 inter-SH2 domain was ligated to the amino terminus of p110, were from Dr. L. T. Williams (San Francisco) [38]. A dominant negative mutant of p85 (∆p85) which lacks a p110 binding site (residues 479-513) was from Dr. M. Sakaue [39].…”

Purification and characterization of sn-1-stearoyl-2-arachidonoylglycerol kinase from pig testes

“…This may be explained by the report that the 80 kDa DAG kinase translocates to the membrane [16,23,26] or cytoskeleton of cells [8], in response to epidermal growth factor stimulation [26]. It has also been shown that protein kinase C isoforms phosphorylate DAG kinase in itro [26,27].…”