Characterization of phospholipase C isozymes in bovine retina: purification of phospholipase C-beta 4.
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Amino Acid Sequence↗
Biomedical subjects
S G Rhee
Publications and source records attributed to S G Rhee.
Inositol polyphosphates are not increased by overexpression of Ins(1,4,5)P3 3-kinase but show cell-cycle dependent changes in growth factor-stimulated fibroblasts.
NIH 3T3 fibroblasts were stably transfected with rat brain inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) 3-kinase to explore the relationship between increased production of Ins(1,3,4,5)P4 and the formation of InsP5 and InsP6. Mass measurements of InsP5 and InsP6 revealed no significant difference between kinase- and vector-transfected fibroblasts. However, such 3-kinase-transfected cells, when labeled with [3H]inositol for 48-72 h, showed lower levels of [3H]InsP5 and [3H]InsP6, as well as [3H]Ins(1,3,4,6)P4 and D/L[3H]Ins(1,4,5,6)P4, than their vector-transfected counterparts. Because Ins(1,4,5)P3 3-kinase-transfected cells grew less rapidly than vector-transfected controls, we determined whether the synthesis of InsP5 and InsP6 was related to a specific phase of the cell cycle. When NIH 3T3 cells prelabeled with [3H]inositol were synchronized by serum deprivation followed by stimulation with platelet-derived growth factor (PDGF), the amounts of labeled InsP5 and InsP6 began to increase only after 12 h of stimulation, when cells entered the S-phase as indicated by increased [3H]thymidine incorporation. The enhanced synthesis of these inositol polyphosphates was preceded by an early increase in Ins(1,4,5)P3 and its metabolites that was no longer evident by the fifth hour of PDGF action. There was also a prominent and biphasic increase in the level of D/L-Ins(1,4,5,6)P4 with an early peak at approximately 3 h and a second rise that paralleled the increases in InsP5 and InsP6. These results indicate that the formation of highly phosphorylated inositols is not tightly coupled to the receptor-mediated formation of Ins(1,4,5)P3 and its metabolites but is mainly determined by other factors that operate at specific points of the cell cycle.
3T3 Cells↗
A thiol-specific antioxidant and sequence homology to various proteins of unknown function.
Yeast and mammalian cells contain a 25 kDa enzyme that protects cellular components against oxidative damage from a system capable of generating reactive sulfur species, but not from a system that generates only reactive oxygen species. Yeast and rat cDNAs corresponding to this thiol-specific antioxidant (TSA) have been cloned and sequenced. Rat TSA is 65.3% identical and 76.2% similar to yeast TSA in amino acid sequence. A search of the GenBank database revealed 12 additional TSA-like proteins, which show sequence identity to rat TSA ranging from 31 to 76%. Except for the AhpC protein identified in Salmonella typhimurium, none of the TSA-like proteins is associated with known cellular functions. AhpC, which exhibits approximately 40% sequence identity to TSA, has been proposed to be a catalytic component of alkyl hydroperoxide reductase. Alignment of rat and yeast TSA with the TSA-like sequences revealed two conserved cysteine residues, one conserved in all 14 sequences and the other in 12 sequences. The most conserved cysteine is located in a well-conserved motif of (hydrophobic residue)6-Pro-non-conserved-residue-Asp-Phe-Thr-Phe-Val-Cys-Pro-Thr-Glu- hydrophobic residue. These results suggest that the TSA-like proteins of previously unknown function may represent a widely distributed family of antioxidants with functions similar to those of TSA and AhpC.
Amino Acid Sequence↗
Activation of phospholipase C-beta 2 mutants by G protein alpha q and beta gamma subunits.
The beta- but not the gamma- and delta-type isozymes of inositol phospholipid-specific phospholipase C (PLC) are activated by G protein alpha q and beta gamma subunits. The beta-type PLC isozymes differ from other isozymes in that they contain a long carboxyl-terminal region downstream of the Y catalytic domain and a region rich in acidic amino acids between the two separated X and Y catalytic domains. To determine the sites on PLC-beta 2 that participate in the interaction of the enzyme with alpha q and beta gamma subunits, we introduced specific truncations and substitutions in the PLC-beta 2 cDNA at positions corresponding to the carboxyl-terminal and acidic amino acid-rich regions, respectively. After transient expression of these cDNA clones in CV-1 cells, the mutant enzymes were partially purified and their capacity to be activated by alpha q and beta gamma subunits determined. Substitution of glutamine residues for three or all seven of a stretch of consecutive glutamic acids in the acidic domain of PLC-beta 2 affected neither alpha q- nor beta gamma-dependent activation significantly. Carboxyl-terminal truncation to residue Gly-934 or to residue Ala-867 resulted in enzymes that were activated by beta gamma but not by alpha q. This result suggests that the carboxyl-terminal region of PLC-beta 2 is required for activation by alpha q, and that beta gamma subunits interact with a different region of the enzyme. Thus, alpha q and beta gamma subunits may independently modulate a single PLC-beta 2 molecule concurrently.
Amino Acid Sequence↗
Purification, molecular cloning, and sequencing of phospholipase C-beta 4.
We have characterized inositol phospholipid-specific phospholipase C (PLC) isozymes in bovine retina. Chromatography of a retinal homogenate on a heparin column partially resolved six peaks of PLC activity, which differed in their relative selectivities for the substrates phosphatidyl 4,5-bisphosphate (PIP2) and phosphatidylinositol (PI). Five of the peaks were shown to correspond to the known PLC isozymes PLC-beta 1, PLC-beta 3, PLC-gamma 1, PLC-delta 1, and PLC-delta 2. PLC-beta 1, PLC-beta 3, PLC-gamma 1, and PLC-delta 1 in the retinal fractions were identified by immunoblotting with isozyme-specific antibodies, and PLC-delta 2 was identified by direct sequencing of tryptic peptides. PLC-gamma 2 and PLC-beta 2 were not detectable by immunoblot analysis. In addition to five of the seven mammalian PLC isozymes identified to date, bovine retina contained a previously unidentified PLC, which exhibited the highest selectivity for PIP2 over PI. The new PLC was purified from a retinal particulate fraction to yield a preparation that contained a major protein band with an apparent molecular mass of 130 kDa on SDS-polyacrylamide gels. Sequence analysis of 12 tryptic peptides derived from the 130-kDa protein suggested that the primary structure of the new PLC is similar to those members of beta-type PLC isozymes, especially to that of PLC-norpA, which was originally identified in Drosophila eye. The new enzyme was thus named PLC-beta 4. A search of a rat brain cDNA library with the polymerase chain reaction and oligonucleotide primers based on common PLC amino acid sequences resulted in the cloning of a rat brain cDNA corresponding to a previously uncharacterized PLC. The cDNA encoded a putative polypeptide of 1176 amino acids, with a calculated molecular mass of 134,532 daltons, that contained the sequences of all 12 tryptic peptides of PLC-beta 4. Furthermore, the deduced amino acid sequence of the encoded protein was more related to PLC-norpA than to any of the three mammalian PLC-beta isozymes. These results suggest that the brain cDNA encodes PLC-beta 4, which is likely a mammalian homolog of PLC-norpA.
Amino Acid Sequence↗
Purification and characterization of recombinant G16 alpha from Sf9 cells: activation of purified phospholipase C isozymes by G-protein alpha subunits.
A cDNA encoding G16 alpha, the alpha subunit of a heterotrimeric guanine nucleotide-binding protein, was expressed in Sf9 cells using recombinant baculovirus. G16 alpha in membrane extracts of Sf9 cells activated phospholipase C-beta 1 (PLC-beta 1) in the presence of guanosine 5'-[gamma-thio]triphosphate; the system could not be activated by Al3+, Mg2+, and F-. The G16 alpha in the cytosolic fraction of Sf9 cells did not stimulate PLC-beta 1. Concurrent expression of the G-protein beta gamma subunit complex increased the amount of G16 alpha in Sf9 cell membranes. The guanosine 5'-[gamma-thio]triphosphate-activated form of G16 alpha was purified from cholate extracts of membranes from cells expressing G16 alpha, and the G-protein beta 2 and gamma 2 subunits. G16 alpha activated PLC-beta 1, PLC-beta 2, and PLC-beta 3 in a manner essentially indistinguishable from that of Gq alpha. G16 alpha-mediated activation of PLC-beta 1 and PLC-beta 3 greatly exceeded that of PLC-beta 2. G16 alpha did not activate PLC-gamma 1 or PLC-delta 1. Thus, two distantly related members of the Gq alpha family, Gq alpha and G16 alpha, have the same ability to activate the known isoforms of PLC-beta.
Aluminum↗
Cloning, sequencing, and mutation of thiol-specific antioxidant gene of Saccharomyces cerevisiae.
We have previously shown that the yeast Saccharomyces cerevisiae contains an antioxidant enzyme that can provide protection against a thiol-containing oxidation system but not against an oxidation system without thiol. This 25-kDa enzyme was thus named thiol-specific antioxidant (TSA). We have now isolated and sequenced a yeast genomic DNA fragment that encodes TSA. Comparison of the predicted amino acid sequence of TSA with those of conventional antioxidant enzymes, including catalases, peroxidases, and superoxide dismutases, revealed no sequence homology. The 195-amino acid TSA sequence contains 2 cysteine residues. Southern blot analysis of petite yeast DNA, studies with protein synthesis inhibitors, and protein immunoblot analyses of cytosolic and mitochondrial proteins suggest that TSA is a cytosolic protein encoded by nuclear DNA (chromosome XIII). The yeast TSA gene was selectively disrupted by homologous recombination. The haploid tsa mutant was viable under air, suggesting that TSA is not essential for cell viability. The growth rates of the tsa mutant and wild-type strains were identical under anaerobic conditions. However, under aerobic conditions, especially in the presence of methyl viologen or a peroxide (t-butyl hydroperoxide or H2O2), the growth rate of the mutant was significantly less than that of wild-type cells. This result suggests that TSA is a physiologically important antioxidant.
Amino Acid Sequence↗
Orthovanadate induces translocation of phospholipase C-gamma 1 and -gamma 2 in permeabilized mast cells.
Rapid activation of phospholipase C (PLC) with a resultant increase in phosphatidylinositol hydrolysis occurs after aggregation of the high affinity receptor for IgE (Fc epsilon RI) on the surface of mast cells. We previously described an increase in PLC activity associated with the particulate fraction of rat basophilic leukemia (RBL) cells after Fc epsilon RI aggregation, and this redistribution of enzyme activity correlated with an increase in immunoreactivity of the gamma 1 isozyme of PLC in the particulate fraction by Western blot analysis (J. Immunol. 148:2194-2200, 1992). We now report that the tyrosine phosphatase inhibitor orthovanadate mimics Fc epsilon RI-mediated activation of PLC-gamma 1 in RBL cells after permeabilization with Staphylococcus aureus alpha-toxin. Orthovanadate treatment of permeabilized cells induced: 1) a large increase in phosphoinositide hydrolysis in endogenously labeled cells; 2) an increase in PLC activity associated with the particulate fraction; and 3) an increase in immunoreactivity of PLC-gamma 1 in Western blots of the particulate fraction. In addition, incubation of RBL cells with either oligomeric IgE or orthovanadate results in the translocation of PLC-gamma 2 from the cytosol to the particulate fraction. All of the above effects were qualitatively similar to those seen after Fc epsilon RI aggregation. These data suggest that translocation and activation of PLC in mast cells are controlled by tyrosine phosphorylation of either the enzyme itself or some regulatory component. The equilibrium can be shifted to the phosphorylated state during either receptor-mediated activation of a tyrosine kinase or by blockade of dephosphorylation.
Animals↗
Purification from Sf9 cells and characterization of recombinant Gq alpha and G11 alpha. Activation of purified phospholipase C isozymes by G alpha subunits.
Members of the Gq alpha subfamily of heterotrimeric guanine nucleotide-binding proteins (G proteins) activate phospholipase C (PLC). The complementary DNAs (cDNAs) for the G protein alpha subunits Gq alpha and G11 alpha were expressed in insect (Sf9) cells using recombinant baculovirus. Active, nonaggregated, and membrane-associated protein was generated only when the alpha subunit cDNA was expressed together with cDNAs encoding G protein beta and gamma subunits. Recombinant alpha subunits (rGq alpha and rG11 alpha) were purified by three-step procedures, as was a PLC-activating alpha subunit(s) endogenous to Sf9 cells. Guanosine 5'-3-(thio)triphosphate (GTP gamma S) activated rGq alpha and rG11 alpha with an apparent K0.5 of 30 microM; similarly high concentrations of the nucleotide were required to observe [35S]GTP gamma S binding to rGq alpha. Activated rGq alpha and rG11 alpha each stimulated all three isoforms of purified PLC-beta with the rank order of potency PLC-beta 1 = PLC-beta 3 > or = PLC-beta 2; both alpha subunits also stimulated PLC-beta 1 and PLC-beta 3 to a much greater extent (10-fold) than they did PLC-beta 2. In contrast, activated rGq alpha and rG11 alpha failed to stimulate either PLC-delta 1 or PLC-gamma 1. Recombinant Gi alpha 1, Gi alpha 2, Gi alpha 3, Go alpha (A), Gs alpha, and Gz alpha all failed to stimulate any of the isoforms of PLC. The apparent affinities of rGq alpha and rG11 alpha for PLC-beta 1 and their capacities to activate the enzyme were similar to values observed for purified brain Gq alpha/11 alpha. Purified brain beta gamma subunits also stimulated the three isoforms of PLC-beta. The capacities of rGq alpha and rG11 alpha to activate PLC-beta 1 and PLC-beta 3 greatly exceeded those of beta gamma, whereas Gq alpha, G11 alpha and beta gamma were roughly equiefficacious with PLC-beta 2; the alpha subunits were more potent than beta gamma in all cases. The effects of alpha and beta gamma together were nonadditive for both PLC-beta 1 and PLC-beta 2. These results demonstrate that Gq alpha and G11 alpha specifically and selectively stimulate beta isoforms of PLC and confirm the idea that these members of the Gq alpha subfamily of G proteins are physiological regulators of this signaling pathway.
Animals↗
Cross-linking of Fc gamma RIIIA on natural killer cells results in tyrosine phosphorylation of PLC-gamma 1 and PLC-gamma 2.
The Fc gamma RIIIA, which is composed of a transmembrane IgG-binding glycoprotein and either a disulfide-linked homodimer (zeta zeta, gamma gamma) or heterodimer (zeta gamma), mediates the antibody-dependent cellular cytotoxicity in NK cells. The role of phospholipase C (PLC) isozymes in Fc gamma RIIIA-mediated signal transduction was investigated. The NK cell line NK3.3 was found to contain PLC-gamma 1 and an especially high concentration of PLC-gamma 2, but PLC-beta 1 and PLC-delta 1 were not detected. Cross-linking of Fc gamma RIIIA on NK3.3 cells induced a rapid phosphorylation of PLC-gamma 1 and PLC-gamma 2 on tyrosine residues. Pretreatment of NK3.3 cells with a tyrosine kinase inhibitor, herbimycin A, prevented the tyrosine phosphorylation of both PLC-gamma 1 and PLC-gamma 2. These results indicate that activation of Fc gamma RIIIA on NK3.3 cells is coupled either directly or indirectly to a nonreceptor tyrosine kinase, which phosphorylates, and thereby activates PLC-gamma 1 and PLC-gamma 2.
Benzoquinones↗
In vitro tyrosine phosphorylation of PLC-gamma 1 and PLC-gamma 2 by src-family protein tyrosine kinases.
The phosphorylation of purified phospholipase C-gamma 1 (PLC-gamma 1) and PLC-gamma 2 by src-family-protein tyrosine kinases (PTKs) P56lck, p53/56lyn, p59hck, p59fyn, and p60src was studied in vitro. All five PTKs phosphorylated PLC-gamma 1 and PLC-gamma 2, suggesting that both PLC-gamma isozymes can be phosphorylated in cells by any of the src-family PTKs in response to the activation of cell surface receptors. Comparison of the in vitro phosphorylation rates revealed no distinct specificity between PLC-gamma 1 and PLC-gamma 2, or between the five PTKs.
Humans↗
Specific expression of inositol 1,4,5-trisphosphate 3-kinase in dendritic spines.
Ultrastructural localization of inositol 1,4,5-trisphosphate 3-kinase (IP3K) in the rat cerebral cortex and hippocampus was studied immunohistochemically. In both regions, the major structure expressing a high level of IP3K was the dendritic spines of pyramidal neurons, where immunoreactivity was associated with the spine apparatuses and plasmalemma. The postsynaptic densities showed the most intense labelling. Taking into account the results of our previous observations, which demonstrated the restricted localization of the enzyme in the dendritic spines of Purkinje and basket cells in cerebellum, IP3K may be localized specifically in dendritic spines in various regions of the central nervous system, and involved in synaptic signal transduction at the spines.
Animals↗
Cloning, sequencing, purification, and Gq-dependent activation of phospholipase C-beta 3.
Six mammalian phospholipase C isozymes (PLC-beta 1, PLC-beta 2, PLC-gamma 1, PLC-gamma 2, PLC-delta 1, and PLC-delta 2) have been identified at both protein and DNA levels. Here, cDNAs corresponding to a previously unidentified PLC isozyme were isolated from a rat thyroid cell FRTL cDNA library. Comparison of the predicted amino acid sequence of this new PLC with other known PLC isozymes revealed a high degree of overall similarity with PLC-beta 1 and PLC-beta 2. Thus, the new PLC was named PLC-beta 3. Comparison with PLC-beta 1 and PLC-beta 2 also revealed that the deduced amino-terminal sequence of PLC-beta 3 was incomplete by 10-20 amino acids. With the use of antibodies raised against synthetic peptides corresponding to PLC-beta 3-specific amino acid sequences, we purified PLC-beta 3 from a rat brain particulate fraction. The purified enzyme exhibited an apparent molecular mass of 152 kDa on SDS-polyacrylamide gels, as compared with 150 and 140 kDa for PLC-beta 1 and PLC-beta 2, respectively. Studies of the activation of PLC-beta isozymes by three alpha subunits of Gq class G proteins, alpha q, alpha 11, and alpha 16 in the presence of guanosine 5-O-(3-thiotriphosphate) (GTP gamma S) revealed that the extent of activation decreased in the order of PLC-beta 1 > or = PLC-beta 3 >> PLC-beta 2 for all three alpha subunits, suggesting a certain degree of specificity in the interaction of Gq alpha subunits with different PLC-beta isozymes.
Amino Acid Sequence↗
Activation of phospholipase C isozymes by G protein beta gamma subunits.
The beta gamma subunits of guanine nucleotide-binding proteins (G proteins) have been shown to activate unidentified phospholipase C (PLC) isozymes (Camps, M., Hou, C., Sidiropoulos, D., Stock, J. B., Jakobs, K. H., and Gierschik, P. (1992) Eur. J. Biochem. 206, 821-831; Blank, J. L., Brattain, K. A., and Exton, J. H. (1992) J. Biol. Chem. 267, 23069-23075). To identify these target PLC isozymes, we measured the effect of bovine brain G protein beta gamma subunits on PLC-beta 1, PLC-beta 2, PLC-beta 3, PLC-gamma 1, and PLC-delta 1 activity by reconstituting purified protein components with lipid vesicles containing [3H]phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2). A nearly saturating concentration of beta gamma produced 2.5-, 4-, 8.5-, and 2-fold increases in PLC-beta 1, PLC-beta 2, PLC-beta 3, and PLC-delta 1 activity, respectively, and no activation of PLC-gamma 1, in the presence of 0.2 microM free Ca2+. The beta gamma-dependent activation of the PLC-beta isozymes does not appear to be the result of increased affinity of the enzymes for Ca2+. The beta gamma-dependent PLC activation could be reversed by addition of the GDP-bound form of the alpha subunit of G(o). The alpha subunits of Gq class G proteins have been shown to activate PLC-beta isozymes in the order of PLC-beta 1 > or = PLC-beta 3 >> PLC-beta 2, which differs from the order of PLC-beta 3 > PLC-beta 2 > PLC-beta 1 for beta gamma-dependent activation. Furthermore, the half-maximal concentration of beta gamma (25 nM) required to activate PLC-beta 3 is much higher than that of Gq alpha subunits (0.6 nM) required to activate PLC-beta 1. These results suggest that the extracellular signals that induce the dissociation of G(o) or Gi, the heterotrimeric G proteins abundant in brain, should enhance the hydrolysis of PtdIns 4,5-P2 in brain primarily through activation of PLC-beta 3 (PLC-beta 2 is not detectable in brain). However, signals that activate the less abundant Gq class heterotrimers should result in the activation primarily of PLC-beta 1 and PLC-beta 3 by the corresponding alpha subunits.
Animals↗
Removal of the carboxyl-terminal region of phospholipase C-beta 1 by calpain abolishes activation by G alpha q.
The 150-kDa phospholipase C (PLC)-beta 1 and three immunologically related proteins with molecular sizes of 140, 100, and 45 kDa were purified from bovine brain extracts. Determination of the amino-terminal amino acid sequence of the 45-kDa protein and immunoblots of the purified proteins with sequence-specific antibodies to peptides corresponding to three different regions of PLC-beta 1 suggest that a single cleavage at the linkage between amino acid residues 880 and 881 of PLC-beta 1 generates the 100- and 45-kDa proteins, which correspond to the amino-terminal and carboxyl-terminal portions, respectively, of PLC-beta 1. The Ca(2+)-dependent protease calpain appears to be responsible for the cleavage of PLC-beta 1; the PLC-beta 1 amino acid sequence contains PEST sequences which are common to proteins susceptible to calpain, and limited proteolysis of purified PLC-beta 1 by calpain generated a 100-kDa protein and a 40-kDa protein that contains the same amino-terminal sequence as the 45-kDa protein. The 140-kDa protein lacks the carboxyl-terminal-most region of PLC-beta 1, but there is no evidence it is derived from PLC-beta 1 by proteolysis. Cleavage of PLC-beta 1 by calpain had no significant effect on catalytic activity measured in the absence of the alpha subunit of the G alpha q but completely abrogated the stimulatory effect of G alpha q. On the other hand, G alpha q activated the 140-kDa enzyme. These results suggest that the region between residue 881 and the most carboxyl-terminal 10 kDa of PLC-beta 1 contains the G alpha q interaction site.
Amino Acid Sequence↗
Phosphoinositide signalling enzymes in rat liver nuclei: phosphoinositidase C isoform beta 1 is specifically, but not predominantly, located in the nucleus.
The presence of phosphoinositide-mobilizing enzymes has been investigated in purified rat liver nuclei by radiolabelling and by probing with antibodies. A Ca(2+)-activated phosphoinositidase C (PIC) is present and was shown immunologically to be the beta 1 isoform. No gamma- or delta-PIC was found. However, only 5% of the total beta 1-PIC isoform is nuclear, with the majority being cytosolic. G alpha q and G alpha 11, the suggested physiological activators of beta 1-PIC, were not present. A PtdIns4P 5-kinase is also present, which immunologically is shown to be the C isoform. All of these nuclear inositide enzymes still remained after the removal of the nuclear envelope with Triton X-100, consistent with the concept of an intranuclear inositide cycle [Divecha, Banfic and Irvine (1991) EMBO. J. 10, 3207-3214].
Animals↗
Developmental profile of inositol 1,4,5-trisphosphate 3-kinase in rat cerebellar cortex: light and electron microscopic immunohistochemical studies.
Developmental expression and intracellular distribution of inositol 1,4,5-trisphosphate 3-kinase in the rat cerebellar cortex were studied immunohistochemically. Immunoreactivity appeared first at postnatal day 1 in the rostral region of the cerebellum and by day 15 had extended throughout the whole cerebellum, being localized in the Purkinje cell layer. Shortly after the expression of the enzyme in each Purkinje cell, the labelling showed a tendency to accumulate in the dendrites in a fine granular pattern. Electron microscopy revealed that immunoreactivity was present in the Purkinje dendritic trunks with accentuation in the distal segments during the early postnatal period, thereafter becoming concentrated in the dendritic spines at later developmental stages. Labelling was associated mainly with the plasmalemma, including the postsynaptic densities and open coated vesicles, and the subplasmalemmal vesicles of the smooth endoplasmic reticulum. Immunoreactivity was also evident in the perisomatic processes of immature Purkinje cells, which are transient projections synapsing with climbing fibers. In developing Purkinje axons, immunoreactivity was accentuated in the distal segments, associated with the plasmalemma and synaptic vesicles. These results suggest that inositol 1,4,5-trisphosphate 3-kinase is involved in the dendritic arborization and subsequent spine synaptogenesis of Purkinje cells, and that the developing presynaptic nerve endings of these cells are another functional site for the enzyme.
Aging↗
Keratinocyte differentiation is associated with changes in the expression and regulation of phospholipase C isoenzymes.
In murine keratinocytes, Ca(++)-induced terminal differentiation is accompanied by a rapid and sustained increase of inositol phosphates and diacylglycerol. Based on Western blotting analysis, basal keratinocytes cultured in 0.05 mM Ca++ medium express phospholipase C (PLC)-gamma 1 predominantly and no detectable PLC-beta 1. Differentiating keratinocytes cultured in 1.4 mM Ca++ express two- to threefold more PLC-gamma 1 protein and PLC-delta 1, but no detectable PLC-beta 1. Although the amount of PLC-gamma 1 and -delta 1 protein increased, PLC-gamma 1 and -delta 1 mRNA decreased in differentiating cells. Thus the sustained rise of PLC activity induced by Ca++ in differentiating keratinocytes may be associated with higher amounts of both PLC-gamma 1 and -delta 1 in maturing cells, determined by a posttranscriptional mechanism. Tyrosine phosphate content in PLC-gamma 1 was low in basal cells and did not change in cells exposed to 1.4 mM Ca++. However, genistein inhibited the increase in PLC activity induced by 1.4 mM Ca++. In contrast, transforming growth factor (TGF)alpha, which stimulates both PLC activity and growth in basal keratinocytes, increased tyrosine phosphorylation of PLC-gamma 1. These results suggest that tyrosine phosphorylation of PLC-gamma 1 by the epidermal growth factor (EGF) receptor is linked to stimulated proliferation, whereas stimulation of PLC activity by Ca++ is linked to keratinocyte differentiation and involves the action of a tyrosine kinase but not tyrosine phosphorylation of PLC-gamma 1. Based on studies using the intracellular free Ca++ chelator BAPTA, a rise in intracellular free Ca++ was not required for stimulation of PLC activity by raising extracellular Ca++. Phorbol esters inhibited PLC stimulation by 1.4 mM Ca++ medium and increased serine phosphorylation of PLC-gamma 1. Exogenous phosphatidylinositol-specific and phosphatidylcholine-specific bacterial PLC also inhibited endogenous inositol phosphate formation and increased endogenous diacylglycerol (DAG). Thus, direct serine phosphorylation of PLC-gamma 1 by protein kinase C is associated with the inhibition of Ca(++)-mediated PLC stimulation. These results show that keratinocytes have multiple mechanisms to regulate PLC activity in response to a specific signal.
Animals↗