A phorbol ester receptor/protein kinase, nPKC eta, a new member of the protein kinase C family predominantly expressed in lung and skin.

The regulation and function of β2-chimaerin, a novel receptor for the phorbol ester tumour promoters and the second messenger DAG (diacylglycerol), is largely unknown. As with PKC (protein kinase C) isoenzymes, phorbol esters bind to β2-chimaerin with high affinity and promote its subcellular distribution. β2-Chimaerin has GAP (GTPase-activating protein) activity for the small GTP-binding protein Rac1, but for not Cdc42 or RhoA. We show that acidic phospholipids enhanced its catalytic activity markedly in vitro, but the phorbol ester PMA had no effect. β2-Chimaerin and other chimaerin isoforms decreased cellular levels of Rac-GTP markedly in COS-1 cells and impaired GTP loading on to Rac upon EGF (epidermal growth factor) receptor stimulation. Deletional and mutagenesis analysis determined that the β2-chimaerin GAP domain is essential for this effect. Interestingly, PMA has a dual effect on Rac-GTP levels in COS-1 cells. PMA increased Rac-GTP levels in the absence of a PKC inhibitor, whereas under conditions in which PKC activity is inhibited, PMA markedly decreased Rac-GTP levels and potentiated the effect of β2-chimaerin. Chimaerin isoforms co-localize at the plasma membrane with active Rac, and these results were substantiated by co-immunoprecipitation assays. In summary, the novel phorbol ester receptor β2-chimaerin regulates the activity of the Rac GTPase through its GAP domain, leading to Rac inactivation. These results strongly emphasize the high complexity of DAG signalling due to the activation of PKC-independent pathways, and cast doubts regarding the selectivity of phorbol esters and DAG analogues as selective PKC activators.

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The complete primary structure of protein kinase C--the major phorbol ester receptor

Science ◽

10.1126/science.3755547 ◽

1986 ◽

Vol 233 (4766) ◽

pp. 853-859 ◽

Cited By ~ 590

Author(s):

P. Parker ◽

L Coussens ◽

N Totty ◽

L Rhee ◽

S Young ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phorbol Ester ◽

Primary Structure ◽

Kinase C ◽

Phorbol Ester Receptor ◽

Complete Primary Structure

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Yeast phenotype classifies mammalian protein kinase C cDNA mutants.

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.4728 ◽

1993 ◽

Vol 13 (8) ◽

pp. 4728-4735 ◽

Cited By ~ 9

Author(s):

H Riedel ◽

L Su ◽

H Hansen

Keyword(s):

Protein Kinase C ◽

Catalytic Activity ◽

Protein Kinase ◽

Phorbol Ester ◽

Colony Size ◽

Receptor Protein ◽

Amino Terminal ◽

Kinase C ◽

Carboxyl Terminal ◽

Pkc Alpha

The phorbol ester receptor protein kinase C (PKC) gene family encodes essential mediators of eukaryotic cellular signals. Molecular dissection of their mechanisms of action has been limited in part by the lack of random mutagenesis approaches and by the complexity of signaling pathways in mammalian cells which involve multiple PKC isoforms. Here we present a rapid screen which permits the quantification of mammalian PKC activity phenotypically in the yeast Saccharomyces cerevisiae. Bovine PKC alpha cDNA is functionally expressed in S. cerevisiae. This results in a phorbol ester response: a fourfold increase in the cell doubling time and a substantial decrease in yeast colony size on agar plates. We have expressed pools of bovine PKC alpha cDNAs mutagenized by Bal 31 deletion of internal, amino-terminal, or carboxyl-terminal sequences and have identified three classes of mutants on the basis of their distinct yeast phenotypes. Representatives of each class were analyzed. An internal deletion of amino acids (aa) 172 to 225 displayed ligand-dependent but reduced catalytic activity, an amino-terminal truncation of aa 1 to 153 displayed elevated and ligand-independent activity, and a carboxyl-terminal 26-aa truncation (aa 647 to 672) lacked activity under any conditions. Additional mutations confirmed the distinct functional characteristics of these classes. Our data show that deletion of the V1 and C1 regions results in elevated basal catalytic activity which is still Ca2+ responsive. Internal deletions in the V2 and C2 regions do not abolish phorbol ester or Ca2+ regulation of PKC activity, suggesting that most of the C2 domain is not essential for phorbol ester stimulation and most of the regulatory domain is dispensable for Ca2+ regulation of PKC activity. These distinct activities od the PKC mutants correlate with a specific and proportional yeast phenotype and are quantified on agar plates by yeast colony size. This provides a phenotypic screen which is suitable to identity rare, randomly altered but active mammalian PKC mutants. It quantifies their catalytic and biological activities in response to PKC activators or inhibitors for a systematic mapping of PKC structure and function or PKC-drug interaction.

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