Koh-ichi Nagata

Phospholipid-mediated signaling in receptor activation of human platelets.

This review will describe the transmembrane signaling pathway involving the phospholipid metabolism in human platelets. However, because of limited space, this will not cover the detailed mechanism of Ca 2+ dynamics, i.e., fluxes through the plasma membrane and intracellular mobilization. Several other aspects of platelet signal transduction have been reviewed

The ram: A novel low molecular weight GTP-binding protein cDNA from a rat megakaryocyte library

A novel low Mr GTP‐binding protein cDNA was isolated from a rat megakaryocyte cDNA library with a synthetic oligonucleotide probe corresponding to an 8‐amino acid sequence specific for c25KG, a GTP‐binding protein previously isolated from human platelet cytosol fraction [(1989) J. Biol. Chem. 264, 17000–17005]. The cDNA has an open reading frame encoding a protein of 221 amino acids with a calculated Mr of 25068. The protein is designated as ram (ras‐related gene from megakaryocyte) protein (ram p25). The amino acid sequence deduced from the ram cDNA contains the consensus sequences for GTP‐binding and GTPase domains. ram p25 shares about 23%, 39% and 80% amino acid homology with the H‐ras, smg25A and c25KG proteins, respectively. The 3.5‐kb ram mRNA was detected abundantly in spleen cells.

Stimulation of arachidonic acid release by guanine nucleotide in saponin-permeabilized neutrophils: Evidence for involvement of GTP-binding protein in phospholipase A2 activation

Addition of a guanine nucleotide analog, guanosine 5-O-(thiotriphosphate) (GTP gamma S)(1-100 microM) induced release of [3H]arachidonic acid from [3H]arachidonate-prelabeled rabbit neutrophils permeabilized with saponin. The chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced arachidonate release was enhanced by GTP gamma S, Ca2+, or their combination. Ca2+ alone (up to 100 microM) did not effectively stimulate lipid turnover. However, the combination of fMLP plus GTP gamma S elicited greater than additional effects in the presence of resting level of free Ca2+. The addition of 100 microM of GTP gamma S reduced the Ca2+ requirement for arachidonic acid liberation induced by fMLP. Pretreatment of neutrophils with pertussis toxin resulted in the abolition of arachidonate release and diacylglycerol formation. Neomycin (1 mM) caused no significant reduction of arachidonate release. In contrast, about 40% of GTP gamma S-induced arachidonate release was inhibited by a diacylglycerol lipase inhibitor, RHC 80267 (30 microM). These observations indicate that liberation of arachidonic acid is mediated by phospholipase A2 and also by phospholipase C/diacylglycerol lipase pathways. Fluoride, which bypasses the receptor and directly activates G proteins, induced arachidonic acid release and diacylglycerol formation. The fluoride-induced arachidonate release also appeared to be mediated by these two pathways. The loss of [3H]arachidonate was seen in phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine. These data indicate that a G protein is involved between the binding of fMLP to its receptor and activation of phospholipase A2, and also that the arachidonic acid release is mediated by both phospholipase A2 and phospholipase C/diacylglycerol lipase.

Stimulation by GTP-binding proteins (Gi, Go) of partially purified phospholipase C activity from human platelet membranes

A phospholipase C exhibiting preferential hydrolytic activity for polyphosphoinositides was partially purified from the deoxycholate extract of human platelet membranes by Q-Sepharose and Heparin-Sepharose column chromatographies. The activity of this purified phospholipase C free of the GTP gamma S-binding activity was stimulated at a similar level by addition of purified rat brain Gi or Go. These results suggest that GTP-binding proteins may interact directly with a solubilized membrane phospholipase C to stimulate its activity.

GTP‐binding proteins in human platelet membranes serving as the specific substrate of islet‐activating protein, pertussis toxin

Two GTP‐binding proteins serving as the specific substrate of islet‐activating protein (IAP), pertussis toxin, were purified from human platelet membranes as heterotrimers with an αβγ‐subunit structure. The α of the major IAP substrate had a molecular mass of 40 kDa and differed from that of Gi 1 or Go previously purified from brain membranes. The partial amino acid sequences of the 40 kDa α completely matched with the sequences which were deduced from the nucleotide sequences of the human Gi 2 α gene. On the other hand, the α of the minor IAP substrate purified from human platelets was about 41 kDa and cross‐reacted with an antibody raised against α of brain Gi 1 (Gi 1 α). These results indicate that the major IAP substrate present in human platelet membranes is a product of the Gi 2 α gene.

Redistribution of phospholipidCa2+-dependent protein kinase in mast cells activated by various agonists

Three types of agonists; receptor-mediated concanavalin A), direct (phorbol ester), and membrane-perturbing (compound 48/80), elicit histamine secretion from rat peritoneal mast cells. We tested whether activation of the mast cells by these agents is accompanied by subcellular redistribution of protein kinase C. Phorbol ester treatment predictably caused a profound decrease of phospholipid/Ca2+-dependent histone kinase activity in the cytosol and a concomitant increase of [3H]PMA-binding capacity in the membrane fraction, in a time- and concentration-dependent manner. Similar, but less marked effects were observed with stimulations by concanavalin A and compound 48/80. When mast cells labeled with [32P] and then stimulated with the agents, phosphorylation of a 50,000-Dalton protein was enhanced in the membrane fraction. These results suggest that protein kinase C may play a role in mast cell activation through phosphorylation of the membrane protein.

Redistribution of protein kinase C in pancreatic acinar cells stimulated with caerulein or carbachol

We examined phospholipid/calcium-dependent protein kinase (protein kinase C) activity and amylase secretion in isolated pancreatic acinar cells, when exposed to caerulein or carbachol. Upon stimulation with 10(-10) M caerulein or 10(-6) M carbachol cytosolic protein kinase C activity was increased in accordance with amylase secretion. Effect of carbachol on increase in membrane-associated protein kinase C activity was maximal at 10(-6) M where the rate of amylase secretion was highest. On the other hand, caerulein showed the maximal secretion of amylase at 10(-9) M, but the activity of the protein kinase C associated with membranes increased progressively with increasing concentration of caerulein. These results indicate different profiles of redistribution of protein kinase C upon stimulation of pancreatic acinar cells with carbachol or caerulein, and they were discussed in terms of amylase secretion.

Protein kinase C isozymes in human megakaryoblastic leukaemia cell line, MEG-01: possible involvement of the isozymes in the differentiation process of MEG-01 cells

The expression of the different protein kinase C (PKC) isozymes in various states of differentiation of the human megakaryoblastic leukaemia cell line MEG‐01 were analysed using thermocycle amplification of mRNA and immunoblotting. MEG‐01 expressed mRNAs of PKCα, ‐βI, ‐βII, ‐δ, ‐ε, ‐η, ‐θ and ‐ζ, but not PKCγ. At the protein molecule level, MEG‐01 was observed to express PKCα, ‐βI, ‐βII, ‐ε, ‐θ and ‐ζ, but lack ‐γ, ‐δ and ‐η. When differentiation of MEG‐01 was induced by 100u2003nM 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA), rapid translocation from cytosol to membrane fraction and down‐regulation of PKCα, ‐ε and ‐θ was observed in 1–2u2003h. On the other hand, PKCβI and ‐βII were observed to translocate not only to the membrane fraction but also to the cytoskeletal fraction in a different manner, and their down‐regulation, especially βII, was very slow. The myristoylated, alanine‐rich C kinase substrate (MARCKS) in the membrane fraction of MEG‐01 cells was observed to decrease gradually throughout the differentiation process. Additionally, time‐course study of TPA treatment indicated that incubation of the cells for 30u2003min is sufficient for differentiation. These results strongly suggest that the activation of PKCα, ‐ε and ‐θ is involved in the initiation of differentiation, and that PKCβI and ‐βII have important roles in the maintenance of differentiation. Although PKCζ was resistant to TPA treatment and its translocation was reduced, the amount of this isozyme in the cytosol fraction decreased throughout the differentiation process.

Low Mr GTP-binding proteins in human platelets: Cyclic AMP-dependent protein kinase phosphorylates m22KG(I) in membrane but not c21KG in cytosol

We have purified and characterized two kinds of GTP-binding proteins with Mr of 22,000 in human platelet membrane (main; m22KG(I), minor; m22KG(II)) (Nagata, K. and Nozawa, Y. (1988) FEBS Lett. 238, 90-94). In this study, the main GTP-binding protein (m22KG(I)) was found to be phosphorylated by cyclic AMP-dependent protein kinase (A-kinase), but not by protein kinase C. About 0.5 mol of phosphate was maximally incorporated into one mol of the protein and this phosphorylation was inhibited in the presence of A-kinase inhibitor. Phosphorylation of m22KG(I) did not alter either its GTP-binding or GTPase activity. When m22KG(I) was incubated alone or in the presence of 100 microM guanosine 5-(3-O-thio)triphosphate (GTP gamma S) and then exposed to A-kinase, no significant changes in the level of phosphorylation were observed. On the other hand, the most abundant GTP-binding protein with Mr of 21,000 (c21KG) in human platelet cytosol, which was identified as a transformation suppressor gene product (rap 1 protein, smg p21 and Krev-1 protein), was not phosphorylated by A-kinase under the same condition. However, c21KG was phosphorylated by A-kinase after pretreatment with alkaline phosphatase.

Differential expression of protein kinase C isozymes and small GTP-bincling proteins during HL60 cell differentiation by retinoic acid and cyclic AMP: Relation with phospholipase D (PLD) activation

The differential expression of protein kinase C (PKC) isozymes and small GTP-binding proteins, and their relation to O2 generation and phospholipase D (PLD) activation were analyzed during the differentiation of human promyelocytic HL60 cells to neutrophil-like cells induced by either retinoic acid (RA) or dibutyryl cyclic AMP (dbcAMP). In response to either one of the inducers, nitroblue tetrazolium (NBT) reduction activity time-dependently increased. Although PLD activity was upregulated by dbcAMP-treatment, only a slight increase was observed in RA-treated cells. Small GTP-binding proteins Rac1, Rap1, and RhoA, which are reported to be implicated in O2- generation or PLD activation, were already expressed in undifferentiated HL60 cells and their significant changes were not detected during differentiation. The mRNAs of the cytosolic components of NADPH oxidase system, p47phox and p67phox, were present in trace amounts in undifferentiated cells. However, they rapidly increased in response to RA or dbcAMP. In response to either RA or dbcAMP, the increases were observed in cPKC isozymes (alpha, beta I, beta II) but not in other subtypes (delta, epsilon, theta, zeta) by both RT-PCR and Western blot analyses. In dbcAMP-treated cells PKC alpha increased remarkably, whereas PKC beta I and beta II mainly elevated in RA-treated cells. These results suggest the possibility that cPKCs are closely related to cell differentiation and that PKC alpha is involved in PLD activation.