scholarly journals Activation of purified human protein kinase C α and β I isoenzymes in vitro by Ca2+, phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate

1993 ◽  
Vol 291 (2) ◽  
pp. 627-633 ◽  
Author(s):  
G Kochs ◽  
R Hummel ◽  
B Fiebich ◽  
T F Sarre ◽  
D Marmé ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Substrate Specificity ◽  
Insect Cells ◽  
Recombinant Baculovirus ◽  
Cellular Expression ◽  
Kinase C ◽  
Infected Insect ◽  
Pkc Alpha

The increasing number of eukaryotic protein kinase C (PKC) isoenzymes which have been described has raised great interest in potential differences in the cellular expression, the mode of activation and the substrate specificity of these isoenzymes. The last two aspects have mostly been studied with isoenzymes purified from rat or bovine brain or from recombinant-baculovirus-infected insect cells. In this study, we have expressed the human PKC isoenzymes alpha and beta I in recombinant-baculovirus-infected insect cells. The isoenzymes were purified to homogeneity by a four-step procedure which included a reversible Ca(2+)-dependent association/dissociation to and from the endogenous membranes of the lysed insect cells. Characterization of the purified enzymes with respect to ATP requirement and substrate specificity, using the epidermal-growth-factor receptor peptide and histone III-S respectively, revealed no isoenzyme-specific differences. Activation by trypsin or Ca2+ and a variety of different phospholipids and phosphoinositides (in a mixed-micellar assay) gave the following results. Proteolytic cleavage of the PKC isoenzymes by trypsin generated fully activated phospholipid-independent PKC beta I, whereas PKC alpha reached only 50% of the activity obtained in the presence of phospholipids. PKC alpha and beta I showed no difference in their dependence on Ca2+, diacylglycerol (DAG) and phosphatidylserine (PS). Replacement of either DAG or PS by phosphatidylglycerol, cardiolipin, phosphatidylcholine and several phosphoinositides revealed that PtdIns(4,5)P2 can act as a PKC activator similar to DAG, whereas PtdIns can substitute for PS as a cofactor of activation. Thus, at least for the PKC isoenzymes alpha and beta I, a combination of PtdIns and PtdIns(4,5)P2 can fully replace PS and DAG in vitro as the classical activators of PKC.

PDGF and IL-1 induce and activate specific protein kinase C isoforms in mesangial cells

1996 ◽  
Vol 271 (1) ◽  
pp. F108-F113 ◽  
Author(s):  
M. B. Ganz ◽  
B. Saksa ◽  
R. Saxena ◽  
K. Hawkins ◽  
J. R. Sedor
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
De Novo ◽  
Mesangial Cells ◽  
De Novo Synthesis ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Alpha Beta ◽  
Pkc Alpha

In vitro and in vivo data suggest a remarkable plasticity in the differentiated phenotype of intrinsic glomerular cells, which after injury express new structures and functions. We have shown that a protein kinase C (PKC) isoform, beta II, is expressed in diseased but not normal glomeruli. Since intrarenal cytokine synthesis has been implicated in the pathogenesis of progressive glomerular injury, we have hypothesized that these mediators induce a change in isoform profile. To test this hypothesis in vitro, we have determined whether platelet-derived growth factor (PDGF) and interleukin-1 (IL-1) alter the expression or activation of PKC isoforms in cultured mesangial cells (MCs). By immunoblot and ribonuclease (RNase) protection assays, both PDGF and IL-1 induce as early as 2 h de novo synthesis of PKC-beta II. Since MCs constitutively express PKC-alpha, -beta I, and -zeta, we also determined whether IL-1 or PDGF alter the activity of these isoforms. PDGF maximally induced translocation of PKC-alpha (10 min), -beta I (90 min), -epsilon (120 min), and -zeta (120 min) from the cytosolic to the membrane fraction. IL-1, in contrast, did not alter the distribution of alpha, beta I, or epsilon at any time measured but did induce PKC-zeta translocation. These data suggest inflammatory mediators regulate PKC isoform activity in diseased glomeruli both by de novo synthesis of unexpressed isoforms and by activation of constitutively expressed PKC isoforms.

scholarly journals The regulatory domain of protein kinase C-ε restricts the catalytic-domain-specificity

1991 ◽  
Vol 276 (1) ◽  
pp. 257-260 ◽  
Author(s):  
C Pears ◽  
D Schaap ◽  
P J Parker
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Catalytic Domain ◽  
Structural Basis ◽  
Regulatory Domain ◽  
Substrate Selection ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Alpha

Protein kinase C (PKC) consists of a family of closely related enzymes that can be divided into two subfamilies (alpha, beta and gamma and delta, epsilon and zeta) on the basis of primary sequence. Functional differences have also been described; thus PKC-alpha, PKC-beta and PKC-gamma readily phosphorylate histone IIIS in vitro, whereas PKC-epsilon will not employ this substrate efficiently. We have previously demonstrated, however, that proteolytic cleavage of PKC-epsilon generates a constitutive kinase activity that is an efficient histone IIIS kinase [Schaap, Hsuan, Totty & Parker (1990) Eur. J. Biochem. 191, 431-435]. In order to investigate the structural basis for this switch in specificity, we have constructed a chimaeric protein containing the regulatory domain of PKC-epsilon fused to the catalytic domain of PKC-gamma. When this is expressed in COS1 cells the chimaeric kinase shows a substrate-specificity similar to that of PKC-epsilon rather than to that of PKC-gamma. This demonstrates a role for the regulatory domain in substrate selection of PKC-epsilon.

scholarly journals Insulin increases mRNA levels of protein kinase C-α and -β in rat adipocytes and protein kinase C-α, -β and -θ in rat skeletal muscle

1995 ◽  
Vol 308 (1) ◽  
pp. 181-187 ◽  
Author(s):  
A Avignon ◽  
M L Standaert ◽  
K Yamada ◽  
H Mischak ◽  
B Spencer ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Mrna Levels ◽  
Rat Adipocytes ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Alpha

Effects of insulin of levels of mRNA encoding protein kinase C (PKC)-alpha, PKC-beta, PKC-epsilon and PKC-theta were examined by ribonuclease protection assay in primary cultures of rat adipocytes in vitro, and in rat adipose tissue and gastrocnemius muscle in vivo. In all cases, insulin increased the levels of PKC-alpha mRNA and PKC-beta mRNA, and, in muscle, insulin also increased the level of PKC-theta mRNA. PKC-epsilon mRNA levels, on the other hand, were not altered significantly. Insulin also stimulated the apparent translocation of PKC-alpha, -beta, -epsilon and -theta, to the membrane fractions of adipocytes, adipose tissue and gastrocnemius muscles, and, in some instances, total PKC levels were diminished, e.g. PKC-alpha and PKC-beta in cultured adipocytes in vitro and/or whole adipose tissue in vivo, and PKC-alpha and PKC-theta in the gastrocnemius muscle. Thus, insulin-induced increases in PKC mRNA may have been partly compensatory in nature to restore PKC levels following translocation and proteolytic losses. However, much more severe depletion of PKC-alpha and PKC-beta by phorbol ester treatment in cultured rat adipocytes in vitro resulted in, if anything, smaller increases in PKC-alpha mRNA and PKC-beta mRNA, and it therefore appears that insulin effects on PKC mRNA levels were not simply due to decreases in respective PKC levels. In addition, effects of insulin, particularly on PKC-beta mRNA, could not be attributed to increased glucose metabolism, which alone decreased PKC-beta mRNA in cultured adipocytes in vitro. We conclude that insulin-induced translocation and degradation of PKC-alpha, PKC-beta and PKC-theta are attended by selective increases in their mRNAs. This mechanism of increasing mRNA may be important in maintaining PKC levels during the continued action of insulin.

scholarly journals Effects of insulin on the translocation of protein kinase C-θ and other protein kinase C isoforms in rat skeletal muscles

1995 ◽  
Vol 308 (1) ◽  
pp. 177-180 ◽  
Author(s):  
K Yamada ◽  
A Avignon ◽  
M L Standaert ◽  
D R Cooper ◽  
B Spencer ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Skeletal Muscles ◽  
Phorbol Esters ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Isoforms ◽  
Pkc Alpha

Protein kinase C (PKC)-theta is a newly recognized major PKC isoform in skeletal muscle. In this study we found that insulin provoked rapid biphasic increases in membrane-associated immunoreactive PKC-theta, as well as PKC-alpha, PKC-beta and PKC-epsilon, in rat soleus muscles incubated in vitro. Effects of insulin on PKC isoforms in the soleus were comparable in magnitude with those of phorbol esters. Increases in membrane-associated PKC-theta, PKC-alpha, PKC-beta and PKC-epsilon were also observed in rat gastrocnemius muscles after insulin treatment in vivo. Our findings suggest that PKC-theta, like other diacylglycerol-sensitive PKC isoforms (alpha, beta and epsilon), may play a role in insulin action in skeletal muscles.

scholarly journals High-efficiency expression of rat protein kinase C-γ in baculovirus-infected insect cells

FEBS Letters ◽  
1990 ◽  
Vol 277 (1-2) ◽  
pp. 15-18 ◽  
Author(s):  
Bernd Fiebich ◽  
Hubert Hug ◽  
Dieter Marmé
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
High Efficiency ◽  
Insect Cells ◽  
Kinase C ◽  
Infected Insect
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