Phosphorylation-dependent protein kinase Dactivation

Abstract

The novel mouse serine-threonine kinase protein kinase D (PKD) is activated in intact Swiss 3T3 cells stimulated by phorbol esters, cell permeant diacylglycerols, bryostatin, neuropeptides and growth factors via a phosphorylation-dependent mechanism requiring protein kinase C (PKC) activity. Structural comparison of the PKD catalytic domain with other kinases reveals a close similarity with MEK family kinases, which are activated upon phosphorylation of key serine and threonine residues in a region termed the activation loop. To study the regulation of PKD, we transfected mutant PKD cDNAs in which putative activation loop serine residues 744 and 748 were mutated to either alanine or glutamic acid into COS-7 cells. Replacement of serines 744 and 748 with alanine prevented activation of the overexpressed PKD form upon phorbol ester treatment of cells, whereas replacement with glutamic acid results in full constitutive activation. Single serine to glutamic acid replacement mutants were partially activated. In vivo 32P-labeling and two-dimensional phosphopeptide mapping of PKD and catalytically inactive PKD mutants at serine 744, 748 or at both residues revealed that phorbol ester-sensitive phosphopeptides could be selectively eliminated from patterns observed as a result of these mutations. Treatment of cells with the PKC inhibitor GFI also prevented the appearance of phosphopeptide spots occuring in response to phorbol ester stimulation. These results provide direct evidence that PKD becomes activated in vivo as a consequence of PKC-mediated phosphorylation of serines 744 and 748. These results support our view of PKD as an important clement in PKC signal transduction.

Sphingolipids in human lens membranes: an update on their composition and possible biological implications

“…It is also known that PCs comprise another source of DAGs. The interactions of DAGs with PKC (Diaz-Laviada et al, 1990;Lee and Severson, 1994;Vernhet et al, 1996;Voziyan et al, 1995) and protein kinase D (Matthews et al, 2000a,b;Rozengurt et al, 1995;Waldron et al, 1999;Waldron and Rozengurt, 2000) contribute to lipid transport from the Golgi apparatus to the plasma membrane and thus cell growth.…”

Subtilase cytotoxin produced by locus of enterocyte effacement‐negative Shiga‐toxigenic Escherichia coli induces stress granule formation

“…For example, PKCδ is upstream of PKD1 in reactive oxygen species-mediated mitochondrial depolarization (Zhang et al, 2015), and PKCδ knockdown effectively attenuates PKD1 activation (Asaithambi et al, 2011). PKCε and PKCη interact and activate PKD1 (Waldron et al, 1999a(Waldron et al, ,1999bBrandlin et al, 2002aBrandlin et al, ,2002bDoppler and Storz, 2007). We provide evidence in this study that depletion of PKCδ suppressed basal phospho-PKD1 (S916) and phospho-(S/T) PKD substrate proteins, which were additionally decreased in the presence of SubAB, and enhanced SubAB-mediated SG formation.…”