No abstract

Kinetic properties of the purified a, fi, and y subspecies of protein kinase C (PKC) to respond to diacylglycerol, phosphatidylserine (PtdSer), and Ca2l were reinvestigated in the presence of several fatty acids. Although responses of these enzyme subspecies to the lipids slightly differed from one another, the reaction velocity of these subspecies was significantly enhanced by synergistic action of diacylglycerol and a cis-unsaturated fatty acid. Arachidonic, oleic, linoleic, linolenic, and docosahexaenoic acids were active in this role, whereas saturated fatty acids such as palmitic and stearic acids were inactive. Elaidic acid was also inactive. In the presence of both PtdSer and diacylglycerol, the cis-unsaturated fatty acids increased further an apparent affinity of PKC to Ca2' and allowed the enzyme to exhibit almost full activation at nearly basal levels of Ca21 concentration. The concentration of fatty acid giving rise to the maximum activation of enzyme was -20-50 ,uM. The result presented herein implies that the receptor-mediated release of unsaturated fatty acids from phospholipids may take part, in synergy with diacylglycerol, in the activation of PKC even when the Ca2+ concentration is low. A possibility arises, then, that the activation of PKC is an integral part of the signal-induced degradation cascade of various membrane phospholipids, which is initiated by the actions of phospholipase C and phospholipase A2.The hydrolysis of phosphatidylinositol, particularly its 4,5-bisphosphate, catalyzed by phospholipase C is generally accepted to be crucially important to initiate signal transduction for eliciting cellular responses (1-3). Recent studies have suggested that receptor-mediated hydrolysis of phosphatidylcholine may also be involved in transmembrane signaling (for a review, see refs. 4 and 5). In fact, it is becoming clear that both phospholipase A2 (6) and phospholipase D (7-11; see ref. 4 for additional references) are activated in a signaldependent manner.Early reports from this laboratory (12, 13) have described that diacylglycerol produced in membranes activates protein kinase C (PKC) in the presence of Ca2' and phospholipids, especially phosphatidylserine (PtdSer). Kinetic analysis has shown that diacylglycerol increases an apparent affinity of the enzyme for Ca2' and PtdSer and thereby activates PKC in the micromolar range of Ca2+ concentrations (13). Subsequent studies in several laboratories (14-21) have found that, in the absence of PtdSer, unsaturated fatty acids such as arachidonic and oleic acids may activate PKC to various degrees, most efficiently activating the y subspecies, and a potential role ofunsaturated fatty acids as second messengers has been postulated. More recently, synergistic action of fatty acids and diacylglycerol for the activation of PKC has been reported (refs. 22-25; also S. G. Chen and K. Murakami, personal communication). Studies on the interaction of fatty acids with diacylglycerol and Ca2l have revealed, however, conflicting results. In some studie...

Two complementary DNA's, encoding the complete sequences of 671 and 673 amino acids for subspecies of rat brain protein kinase C, were expressed in COS 7 cells. The complementary DNA sequence analysis predicted that the two enzymes are derived from different ways of splicing and differ from each other only in the short ranges of their carboxyl-terminal regions. Both enzymes showed typical characteristics of protein kinase C that responded to Ca2+, phospholipid, and diacylglycerol. The enzymes showed practically identical physical and kinetic properties and were indistinguishable from one of the several subspecies of protein kinase C that occurs in rat brain but not in untransfected COS 7 cells. Partial analysis of the genomic structure confirmed that these two subspecies of protein kinase C resulted indeed from alternative splicing of a single gene.

ABSTRACT2-Lysophosphatidylcholine (lysoPtdCho), a product of the hydrolysis of phosphatidylcholine catalyzed by phospholipase A2, greatly potentiates the activation of human resting T lymphocytes that is induced by a membranepermeant diacylglycerol plus a calcium ionophore, as determined by the expression of the a subunit of the interleukin 2 receptor and thymidine incorporation into DNA. LysoPtdCho per se is inactive unless both diacylglycerol and a calcium ionophore are present. This effect of lysoPtdCho is also observed when diacylglycerol is replaced by a tumor-promoting phorbol ester. Other lysophosphatides including lysophosphatidylserine, lysophosphatidylinositol, and lysophosphatidic acid are inert except for lysophosphatidylethanolamine, which is far less effective than IysoPtdCho. Tracer experiments with radioactive choline indicate that, when T lymphocytes are stimulated with an antigenic signal, lysoPtdCho is indeed produced in a time-dependent fashion, although the concentration of this lysophospholipid accumulated remains to be quantitated. It suggests that phospholipase A2 is directly involved in the signal transduction pathway through protein kinase C to induce long-term cellular responses.A single dose of a tumor-promoting phorbol ester, together with a calcium ionophore, can induce T-lymphocyte activation, as determined by secretion of interleukin 2, expression of the a subunit of the interleukin 2 receptor (IL-2Ra), and incorporation of thymidine into DNA (for a review, see ref.1). On the other hand, a single dose of a membrane-permeant diacylglycerol (DAG) is normally insufficient to induce cell activation due to its rapid metabolism within the cell, and it is known that sustained activation of protein kinase C (PKC) by a large dose or repeated doses of a membrane-permeant DAG is essential to induce activation of T lymphocytes (2, 3). The formation of DAG from receptor-mediated hydrolysis of inositol phospholipids is, however, normally transient, and recent evidence strongly suggests that, at relatively later phases in cellular responses, DAG is produced from signalinduced breakdown of phosphatidylcholine (PtdCho), which is initiated presumably by the activation of phospholipase D (for reviews, see refs. 4 and 5). Previous reports from this laboratory (6, 7) suggest that cis-unsaturated fatty acids greatly enhance PKC activation when DAG is available. It has also been briefly described that 2-lysophosphatidylcholine (lysoPtdCho), the other product of receptor-mediated hydrolysis of PtdCho by phospholipase A2, synergizes with a membrane-permeant DAG to activate human resting T lymphocytes (8). In fact, activation of both phospholipase C and phospholipase A2 by a single agonist has been reported by Axelrod and coworkers (9). The receptor-mediated degradation of various membrane phospholipids, therefore, may be important in causing cellular responses such as cell proliferation and differentiation. The studies presented herein were undertaken to explore further the detailed kinects of the lysoPtd...

Rat brain protein kinase C purified to apparent homogeneity [(1986) Biochem. Biophys. Res. Commun. 135, 6366431 was resolved into three distinct fractions, type I, II and III, upon chromatography on a hydroxyapatite column connected to high-performance liquid chromatography. Comparison of each fraction with the four subspecies of protein kinase C, that were separately expressed in COS cells transfected by the respective cDNAs, a, /II, j3II an d y, identified the primary structures of these three fractions of protein kinase C. Type I corresponded to the enzyme encoded by the y-sequence; type II was a mixture of the two subspecies determined by the /?I-and /JR-sequences; and type III had the structure encoded by the a-sequence. The structures and properties of these subspecies of protein kinase C were similar to each other.Protein kinase C; cDNA; (COS cell)

Although a single dose of phorbol 12-myristate 13-acetate (PMA) allowed HL-60 cells to differentiate to macrophages, a single dose of membrane-permeant diacylglycerol (DAG), 1,2-dioctanoylglycerol (1,2-DiC8), was normally isufcient to differentiate these cells. These cells metabolized 1,2-DiCs very rapidly, and 1,2-DiC8 available to protein kinase C (PKC) activation was removed from the incubation medium at a rate proportional to cell density. However, increasing the duration of exposure of HL-60 cells to this DAG either by its repeated addition or by decreasing the cell density greatly enhanced their differentiation to macrophages as Measured by CD11b expression. During this differentiation induced by DAG, neither measurable translocation nor depletion (down-regulation) of PKC was observed. When the cells were exposed to PMA, on the other hand, some PKC subspecies were instantaneously translocated to membranes and subsequently disappeared very quickly, whereas the a-subspecies was decreased to the level of w6O% of the resting cell, but thereafter its activity was maintained at a nearly constant level in membranes. After -4 hr, the PKC subspecies, once depleted, reappeared gradually in the membrane fraction.The results suggest that sa activation of PKC is essential to differentiation of HL-60 cells to macrophages, and depletion of the enzyme is not needed. Perhaps translocation of PKC represents an extreme state of the active form of the enzyme, which may result from PMA action, and the a-subspecies presumably-plays a key role in HL-60 cell differentiation.The HL-60 cell line is frequently used as a model system for investigation of the mechanism of cell differentiation, since retinoic acid and several other chemicals lead this cell to differentiate to a granulocyte, whereas phorbol 12-myristate

Elucidation of the complete sequences of four cDNA clones (a, @I, fl1, and y) of the rat brain protein kinase C family has revealed their common structure composed of a single polypeptide chain with four constant (C,-C,) and five variable (VI-V,) regions. Although these sequences are highly homologous and closely related to one another Vj-, Vd-, and Vs-regions of y-subspecies are slightly bigger than the corresponding regions of the other three subspecies. The first constant region, C,, contains a tandem repeat of cysteine-rich sequence (6, total 12 cysteine residues). The third constant region, Cj, has an ATP-binding sequence which is found in many protein kinases. In adult rat whole brain, the relative activities of tl-, /3I-, /III-, and y-subspecies are roughly 16, 8, 55, and 21%, respectively. y-Subspecies is expressed after birth apparently only in the central nervous tissue, implying its role in the regulation of specific neuronal functions.Protein kinase C: Complementary DNA

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].