Phosphorylation sites of protein kinase C delta in H2O2-treated cells and its activation by tyrosine kinase in vitro.

Protein kinase C delta (PKC delta) is normally activated by diacylglycerol produced from receptor-mediated hydrolysis of inositol phospholipids. On stimulation of cells with H(2)O(2), the enzyme is tyrosine phosphorylated, with a concomitant increase in enzymatic activity. This activation does not appear to accompany its translocation to membranes. In the present study, the tyrosine phosphorylation sites of PKC delta in the H(2)O(2)-treated cells were identified as Tyr-311, Tyr-332, and Tyr-512 by mass spectrometric analysis with the use of the precursor-scan method and by immunoblot analysis with the use of phosphorylation site-specific antibodies. Tyr-311 was the predominant modification site among them. In an in vitro study, phosphorylation at this site by Lck, a non-receptor-type tyrosine kinase, enhanced the basal enzymatic activity and elevated its maximal velocity in the presence of diacylglycerol. The mutation of Tyr-311 to phenylalanine prevented the increase in this maximal activity, but replacement of the other two tyrosine residues did not block such an effect. The results indicate that phosphorylation at Tyr-311 between the regulatory and catalytic domains is a critical step for generation of the active PKC delta in response to H(2)O(2).

The protein kinase C family and lipid mediators for transmembrane signaling and cell regulation.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan.

Regulation of nuclear translocation of forkhead transcription factor AFX by protein kinase B.

The regulation of intracellular localization of AFX, a human Forkhead transcription factor, was studied. AFX was recovered as a phosphoprotein from transfected COS-7 cells growing in the presence of FBS, and the phosphorylation was eliminated by wortmannin, a potent inhibitor of phosphatidylinositol (PI) 3-kinase. AFX was phosphorylated in vitro by protein kinase B (PKB), a downstream target of PI 3-kinase, but a mutant protein in which three putative phosphorylation sites of PKB had been replaced by Ala was not recognized by PKB. In Chinese hamster ovary cells (CHO-K1) cultured with serum, the AFX protein fused with green fluorescence protein (AFX-GFP) is localized mainly in the cytoplasm, and wortmannin induced transient nuclear translocation of the fusion protein. The AFX-GFP mutant in which all three phosphorylation sites had been replaced by Ala was detected exclusively in the cell nucleus. AFX-GFP was in the nucleus when the cells were infected with an adenovirus vector encoding a dominant-negative form of either PI 3-kinase or PKB, whereas the fusion protein stayed in the cytoplasm when the cells expressed constitutively active PKB. In CHO-K1 cells expressing AFX-GFP, DNA fragmentation was induced by the stable PI 3-kinase inhibitor LY294002, and the expression of the active form of PKB suppressed this DNA fragmentation. The phosphorylation site mutant of AFX-GFP enhanced DNA fragmentation irrespective of the presence and absence of PI 3-kinase inhibitor. These results indicate that the nuclear translocation of AFX is negatively regulated through its phosphorylation by PKB.

Requirement of GM2 ganglioside activator for phospholipase D activation.

Sequence analysis of a heat-stable protein necessary for the activation of ADP ribosylation factor-dependent phospholipase D (PLD) reveals that this protein has a structure highly homologous to the previously known GM2 ganglioside activator whose deficiency results in the AB-variant of GM2 gangliosidosis. The heat-stable activator protein indeed has the capacity to enhance enzymatic conversion of GM2 to GM3 ganglioside that is catalyzed by beta-hexosaminidase A. Inversely, GM2 ganglioside activator purified separately from tissues as described earlier [Conzelmann, E. & Sandhoff, K. (1987) Methods Enzymol. 138, 792-815] stimulates ADP ribosylation factor-dependent PLD in a dose-dependent manner. At higher concentrations of ammonium sulfate, the PLD activator protein apparently substitutes for protein kinase C and phosphatidylinositol 4,5-bisphosphate, both of which are known as effective stimulators of the PLD reaction. The mechanism of action of the heat-stable PLD activator protein remains unknown.

Activation of protein kinase C by tyrosine phosphorylation in response to H2O2.

Protein kinase C (PKC) isoforms, alpha, betaI, and gamma of cPKC subgroup, delta and epsilon of nPKC subgroup, and zeta of aPKC subgroup, were tyrosine phosphorylated in COS-7 cells in response to H2O2. These isoforms isolated from the H2O2-treated cells showed enhanced enzyme activity to various extents. The enzymes, PKC alpha and delta, recovered from the cells were independent of lipid cofactors for their catalytic activity. Analysis of mutated molecules of PKC delta showed that tyrosine residues, which are conserved in the catalytic domain of the PKC family, are critical for PKC activation induced by H2O2. These results suggest that PKC isoforms can be activated through tyrosine phosphorylation in a manner unrelated to receptor-coupled hydrolysis of inositol phospholipids.

Taxonomy and function of C1 protein kinase C homology domains.

C1 domains are compact alpha/beta structural units of about 50 amino acids which tightly bind two zinc ions. These domains were first discovered as the loci of phorbol ester and diacylglycerol binding to conventional protein kinase C isozymes, which contain 2 C1 domains (C1A and C1B) in their N-terminal regulatory regions. We present a comprehensive list of 54 C1 domains occurring singly or doubly in 34 different proteins. Many C1 domains and C1 domain-containing proteins bind phorbol esters, but many others do not. By combining analysis of 54 C1 domain sequences with information from previously reported solution and crystal structure determinations and site-directed mutagenesis, profiles are derived and used to classify C1 domains. Twenty-six C1 domains fit the profile for phorbol-ester binding and are termed "typical." Twenty-eight other domains fit the profile for the overall C1 domain fold but do not fit the profile for phorbol ester binding, and are termed "atypical." Proteins containing typical C1 domains are predicted to be regulated by diacylglycerol, whereas those containing only atypical domains are not.

Ca2+ -independent cytosolic phospholipase A in HL-60 cells differentiating to granulocytes.

The release of various fatty acids (FAs) from permeabilized HL-60 cells, predominantly oleic acid (OA) rather than arachidonic acid, was greatly enhanced by GTP-gamma-S and vanadate [Tsujishita, Y., Asaoka, Y. and Nishizuka, Y., Proc. Natl. Acad. Sci. USA 91 (1994) 6274-62781. The present study shows that phospholipase A (A2/A1) activity which cleaves the acyl group from both sn-2 and sn-1 positions of phosphatidylethanolamine (PtdEtn) is increased in HL-60 cells during differentiation to granulocyte-like cells. This enzyme does not require Ca2+ and releases various FAs, preferentially OA from PtdEtn and, to lesser extent, from lysoPtdEtn. Other phospholipids including phosphatidylcholine and phosphatidic acid serve as very poor substrates. Although further studies are necessary to show the direct link of this enzyme activation to receptor stimulation, the results described here imply that this enzyme is responsible for the release of various FAs, particularly OA, from permeabilized HL-60 cells.

Reconstitution of GTP-gamma-S-dependent phospholipase D activity with ARF, RhoA, and a soluble 36-kDa protein.

For activation of kidney membrane phospholipase D (PLD), cytosol is absolutely needed in addition to GTP-gamma-S. The active component of cytosol consists of three protein factors: ADP-ribosylation factor, RhoA, and a soluble 36-kDa protein. Any combination of these two factors synergistically activates PLD to some extent, but the presence of the three factors causes full activation. The 36-kDa protein is stable at 60 degrees C but inactivated at 80 degrees C for 10 min. Tissue distribution of the 36-kDa protein roughly coincides with that of PLD, suggesting physiological relevance of the protein in the regulation of PLD.

Mammalian phospholipase D: phosphatidylethanolamine as an essential component.

Bovine kidney phospholipase D (PLD) was assayed by measuring the formation of phosphatidylethanol from added radioactive phosphatidylcholine (PtdCho) in the presence of ethanol, guanosine 5'-[gamma-thio]triphosphate, ammonium sulfate, and cytosol factor that contained small GTP-binding regulatory proteins. The PLD enzyme associated with particulate fractions was solubilized by deoxycholate and partially purified by chromatography on a heparin-Sepharose column. This PLD preferentially used PtdCho as substrate. After purification, the enzyme per se showed little or practically no activity but required an additional factor for the enzymatic reaction. This factor was extracted with chloroform/methanol directly from particulate fractions of various tissues, including kidney, liver, and brain, and identified as phosphatidylethanolamine (PtdEtn), although this phospholipid did not serve as a good substrate. Plasmalogen-rich PtdEtn, dioleoyl-PtdEtn, and L-alpha-palmitoyl-beta-linoleoyl-PtdEtn were effective, but dipalmitoyl-PtdEtn was inert. Sphingomyelin was 30% as active as PtdEtn. The results suggest that mammalian PLD reacts nearly selectively with PtdCho in the form of mixed micelles or membranes with other phospholipids, especially PtdEtn.

Mammalian phospholipase D: activation by ammonium sulfate and nucleotides.

Phospholipase D (PLD) associated with the rat kidney membrane was activated by guanine 5'-[gamma-thio]triphosphate and a cytosol fraction that contained ADP-ribosylation factor. When assayed by measuring the phosphatidyl transfer reaction to ethanol with exogenously added radioactive phosphatidylcholine as substrate, the PLD required a high concentration (1.6 M) of ammonium sulfate to exhibit high enzymatic activity. Other salts examined were far less effective or practically inactive, and this dramatic action of ammonium sulfate is not simply due to such high ionic strength. Addition of ATP but not of nonhydrolyzable ATP analogue adenosine 5'-[beta, gamma-imido]diphosphate further enhanced the PLD activation approximately equal to 2- to 3-fold. This enhancement by ATP needed cytosol, implying a role of protein phosphorylation. A survey of PLD activity in rat tissues revealed that, unlike in previous observations reported thus far, PLD was most abundant in membrane fractions of kidney, spleen, and liver in this order, and the enzymatic activity in brain and lung was low.

The protein kinase C isoforms leading to MAP-kinase activation in CHO cells.

Stimulation of CHO cells with phorbol ester caused transient activation of mitogen-activated protein kinase (MAP-kinases with 42 and 44 kDa). A similar treatment of CHO cells which overexpress a massive amount of the alpha- or delta-isoform of protein kinase C (PKC) resulted in the prolonged activation of MAP-kinase and eventually in the appearance of mostly dikaryotic, sometimes tetrakaryotic cells. The results suggest that the delta-isoform, as well as the alpha-isoform, has a potential to cause MAP kinase activation. Unusual activation of the PKC/MAP-kinase pathway, however, may lead to abnormal cytokinesis.

Protein kinase C and lipid signaling for sustained cellular responses.

Since the second messenger role was proposed for the products of inositol phospholipid hydrolysis, considerable progress has been made in our understanding of the biochemical mechanism of the intracellular signaling network. It is now becoming evident that stimulation of a cell surface receptor initiates a degradation cascade of various membrane lipid constituents. Many of their metabolites have potential to induce, intensify, and prolong the activation of protein kinase C that is needed for sustained cellular responses.

Lipid mediators and protein kinase C for intracellular signalling.

1. The stimulation of a cell surface receptor initiates a degradation cascade of various membrane lipid constituents. 2. Many of their metabolites have potentials to induce, intensify and prolong the activation of protein kinase C that is needed for sustained cellular responses.

Regulation of phospholipase A2 in human leukemia cell lines: its implication for intracellular signaling.

Permeabilized human leukemia HL-60 and U-937 cells suspended in an acidic or alkaline medium release various unsaturated fatty acids, most abundantly oleic and arachidonic acids. Concomitant production of lysophospholipids suggests that phospholipases A2 play a major role in this fatty acid release reaction. The fatty acid release at acidic conditions depends on the intracellular Ca2+ concentrations at the 10(-8)-10(-7) M range and is enhanced by membrane-permeant diacylglycerols, although this enhancement seems independent of protein kinase C activation. On the other hand, the fatty acid release at alkaline conditions is potentiated by vanadate, and this potentiation is counteracted by genistein, suggesting a role of tyrosine phosphorylation in this release reaction. GTP[gamma S], an activator of G proteins, greatly enhances the fatty acid release. Aluminum fluoride, another activator of heterotrimeric G proteins, also greatly potentiates this release reaction. Phorbol ester increases the fatty acid release at alkaline conditions, to some extent, whereas it counteracts the vanadate-induced potentiation of fatty acid release. The results imply that several phospholipases A2 are coupled to receptors for their activation, thereby functioning in the transmembrane control of cellular events.

Lipid mediators and protein kinase C activation for the intracellular signaling network.

Upon stimulation of a cell surface receptor, a membrane phospholipid degradation cascade is often induced through the activation of several phospholipases, yielding various lipid metabolites such as diacylglycerol, free fatty acids, lysophospholipids and phosphatidic acid. Several isoforms of protein kinase C are each activated distinctly by various combinations of the lipid metabolites, presumably in different subcellular compartments. The pivotal role of this enzyme family in the intracellular signaling network is beginning to emerge.

Potential role of phospholipase A2 in HL-60 cell differentiation to macrophages induced by protein kinase C activation.

2-Lysophosphatidylcholine and cis-unsaturated fatty acids such as linoleic and linolenic acids, which are the products of the hydrolysis of phosphatidylcholine catalyzed by phospholipase A2 (EC 3.1.1.4), significantly potentiate the differentiation of HL-60 cells to macrophages that is induced by either a membrane-permeant diacylglycerol or a phorbol ester. The cell differentiation was assayed by measuring the expression of CD11b, one of the cell surface markers of macrophages, and also by the appearance of phagocytic activity. Snake venom phospholipase A2 added directly to the cells is also active for this potentiation. Neither lysophosphatidylcholine, fatty acid, nor phospholipase A2 is active unless a membrane-permeant diacylglycerol or a phorbol ester is present. The results presented provide further evidence that activation of phospholipase A2 may be intimately related to the signal transduction pathway through protein kinase C.

Potentiation of diacylglycerol-induced activation of protein kinase C by lysophospholipids. Subspecies difference.

Lysophospholipid, particularly 2-lysophosphatidylcholine (lysoPtdCho), significantly potentiates the diacylglycerol (DAG)-induced activation of protein kinase C (PKC) in vitro. LysoPtdCho shows no effect, unless DAG and phosphatidylserine (PtdSer) are present. This lysoPtdCho action also depends on its own as well as on Ca2+ concentration. At physiological Ca2+ concentrations, the activation of the alpha-, beta-, and gamma-subspecies (cPKC) is enhanced by lysoPtdCho in the 10(-6) M range, but inversely inhibited in the 10(-5) M range. The delta- and epsilon-subspecies (nPKC), which are enzymatically insensitive to Ca2+, are mostly inhibited by lysoPtdCho at its low concentrations. The enhancement of cPKC activation by lysoPtdCho is due to the increase in an apparent affinity of the enzyme for PtdSer but not for DAG. The results may account, at least in part, for the previous observations made with intact cell systems that lysoPtdCho significantly potentiates the DAG-induced cellular responses such as T-lymphocyte activation and HL-60 cell differentiation [(1992) Trends Biochem. Sci. 17, 414-417].