Protein kinase C, an enzyme that is activated by the receptor-mediated hydrolysis of inositol phospholipids, relays information in the form of a variety of extracellular signals across the membrane to regulate many Ca2+-dependent processes. At an early phase of cellular responses, the enzyme appears to have a dual effect, providing positive forward as well as negative feedback controls over various steps of its own and other signaling pathways, such as the receptors that are coupled to inositol phospholipid hydrolysis and those of some growth factors. In biological systems, a positive signal is frequently followed by immediate negative feedback regulation. Such a novel role of this protein kinase system seems to give a logical basis for clarifying the biochemical mechanism of signal transduction, and to add a new dimension essential to our understanding of cell-to-cell communication.

https://science.sciencemag.org/content/sci/233/4761/305.full.pdf

Studies and perspectives of protein kinase C

In recent years, members of the protein kinase family have been discovered at an accelerated pace. Most were first described, not through the traditional biochemical approach of protein purification and enzyme assay, but as putative protein kinase amino acid sequences deduced from the nucleotide sequences of molecularly cloned genes or complementary DNAs. Phylogenetic mapping of the conserved protein kinase catalytic domains can serve as a useful first step in the functional characterization of these newly identified family members.

http://science.sciencemag.org/content/sci/241/4861/42.full.pdf

The protein kinase family: conserved features and deduced phylogeny of the catalytic domains.

Hydrolysis of inositol phospholipids by phospholipase C is initiated by either receptor stimulation or opening of Ca2+ channels. This was once thought to be the sole mechanism to produce the diacylglycerol that links extracellular signals to intracellular events through activation of protein kinase C. It is becoming clear that agonist-induced hydrolysis of other membrane phospholipids, particularly choline phospholipids, by phospholipase D and phospholipase A2 may also take part in cell signaling. The products of hydrolysis of these phospholipids may enhance and prolong the activation of protein kinase C. Such prolonged activation of protein kinase C is essential for long-term cellular responses such as cell proliferation and differentiation.

https://science.sciencemag.org/content/sci/258/5082/607.full.pdf

Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C

Protein kinase C is now known to be a large family of proteins, with multiple subspecies that have subtle individual enzymological characteristics. Some members of the family exhibit distinct patterns of tissue expression and intracellular localization; different kinases probably have distinct functions in the processing and modulation of a variety of physiological and pathological responses to external signals.

The molecular heterogeneity of protein kinase C and its implications for cellular regulation

A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002).

Cloning and expression of multiple protein kinase C cDNAs

Sphingosine, sphinganine, and other long-chain (sphingoid) bases inhibit protein kinase C in vitro and block cellular responses to agonists that are thought to act via this enzyme. To gain further insight into the mechanism of this inhibition, a series of long-chain analogues differing in alkyl chain length (11-20 carbon atoms), stereochemistry, and headgroup were examined for (a) inhibition of protein kinase C activity in vitro, (b) the neutrophil respiratory burst in response to phorbol myristate acetate (PMA), (c) the PMA-induced differentiation of HL-60 cells, and (d) the growth of Chinese hamster ovary cells. In every instance, the effects were maximal with the 18-carbon homologues, which are the same length as the predominant naturally occurring long-chain base (sphingosine). The lower potency of the shorter chain homologues was partially due to decreased uptake by cells. Small differences were obtained with the four stereoisomers of sphingosine (i.e., D and L forms of erythro- and threo-sphingosine), with N-methyl derivatives of the different sphingosine homologues, and with simpler alkylamines (e.g., stearylamine). The potency of the different headgroup analogues may be affected by the degree of protonation at the assay pH. The pKa of sphingosine was measured to be 6.7; the pKa varied among the analogues. These findings establish that the major structural features required for inhibition of protein kinase C and cellular processes dependent on this enzyme are the presence of a free amino group and an aliphatic side chain and that other groups have more subtle effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural requirements for long-chain (sphingoid) base inhibition of protein kinase C in vitro and for the cellular effects of these compounds

The specificity of protein kinase C activation by sn-1,2-diacylglycerols and analogues was investigated by using a Triton X-100 mixed micellar assay [Hannun, Y. A., Loomis, C. R. & Bell, R. M. (1985) J. Biol. Chem. 260, 10039-10043]. Analogues containing acyl or alkyl chains eight carbons in length were synthesized because sn-1,2-dioctanoylglycerol is an effective cell-permeant activator of protein kinase C. These analogues were tested as activators and antagonists of rat brain protein kinase C to determine the exact structural features important for activity. The analogues established that activation of protein kinase C by diacylglycerols is highly specific. Several analogues established that both carbonyl moieties of the oxygen esters are required for maximal activity and that the 3-hydroxyl moiety is also required. None of the analogues were antagonists. These data, combined with previous investigations, permitted formulation of a model of protein kinase C activation. A three-point attachment of sn-1,2-diacylglycerol to the surface-bound protein kinase C-phosphatidylserine-Ca2+ complex is envisioned to cause activation. Direct ligation of diacylglycerol to Ca2+ is proposed to be an essential step in the mechanism of activation of protein kinase C.

Specificity and mechanism of protein kinase C activation by sn-1,2-diacylglycerols.

Publisher Summary The hypothesis that sn-l, 2-diacylglycerols function as the intracellular second messengers of growth factors, hormones, and neurotransmitters has gained wide acceptance. This method has been used successfully to measure basal and stimulated diacylglycerol levels in a variety of tissues and cells, including human platelets, rat hepatocytes, sis- and ras-transformed Normal Rat Kidney (NRK) cells, a human leukemia cell line (HL60), and human neutrophils. sn-l, 2-Dioleoylglycerol is prepared from sn-l,2-dioleoyl-glycerol-3-phosphocholine (Avanti) by phospholipase digestion, purified by extraction in ether, and quantitated by ester analysis using cholesteryl acetate as a standard. Bovine heart cardiolipin in chloroform is from Avanti and should be stored under N 2 after opening. This chapter discusses the preparation of membranes containing diacylglycerol kinase. With the recent explosion of interest in diacylglycerols as the in vitro activator of the Ca 2+ -activated phospholipid dependent protein kinase (protein kinase C) and the implication that diacylglycerols function as intracellular second messengers, this assay method provides a useful tool to explore the role of diacylglycerols as the second messengers of neurotransmitters, hormones, and growth factors in a number of biological settings.

Quantitative measurement of sn-1,2-diacylglycerols.

Compositions for inhibiting protein kinase C, comprising an inhibitory amount of a compound having formula (I), wherein Q is a hydrophobic group; wherein X is -CH2-CH2- or -CH=CH-, which may be substituted by one or more halogens or C1-C3 alkyl groups, wherein Y is (II), (III), (IV), (V), or (VI), wherein W is a halogen; wherein R1 and R2 may be the same or different and are selected from hydrogen, lower alkyl groups having from 1 to 7 carbon atoms, aralkyl, and aryl groups, and wherein Z is a phosphate or an organic group, and a pharmaceutically acceptable carrier material; and a method for inhibiting protein kinase C using such compositions.

Carson R. Loomis

Papers

Cloning and expression of multiple protein kinase C cDNAs

Structural requirements for long-chain (sphingoid) base inhibition of protein kinase C in vitro and for the cellular effects of these compounds

Specificity and mechanism of protein kinase C activation by sn-1,2-diacylglycerols.

Quantitative measurement of sn-1,2-diacylglycerols.

Inhibition of protein kinase c by long-chain bases