Effects of protein kinase C activators on germinal vesicle breakdown and polar body emission of mouse oocytes

Protein phosphorylation mediated by cAMP-dependent protein kinase is instrumental in maintaining meiotic arrest of mouse oocytes. To assess whether protein phosphorylation mediated by calcium/phospholipid-dependent protein kinase (protein kinase C) might also inhibit the resumption of meiosis, we treated oocytes with activators of this enzyme. The active phorbol esters 12-O-tetra-decanoyl phorbol-13-acetate (TPA) and 4 beta-phorbol 12,13-didecanoate (4 beta-PDD) inhibited germinal vesicle breakdown (GVBD), as did a more natural activator of protein kinase, C, sn-1,2-dioctanoylglycerol (diC8). An inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), did not inhibit GVBD. We then examined whether protein kinase C activators inhibit a step in the cAMP-modulated pathway that regulates resumption of meiosis. TPA did not inhibit the maturation-associated decrease in oocyte cAMP. Microinjected heat-stable protein inhibitor of cAMP-dependent protein kinase failed to induce GVBD in the presence of TPA. Both TPA and diC8 partially inhibited specific changes in oocyte phosphoprotein metabolism that are tightly correlated with resumption of meiosis; these agents also induced the apparent phosphorylation of specific oocyte proteins. These results suggest that protein kinase C activators may inhibit resumption of meiosis by acting distal to a decrease in cAMP-dependent protein kinase activity, but prior to changes in oocyte phosphoprotein metabolism that are presumably required for resumption of meiosis. Finally, we compared the effects of db-cAMP and protein kinase C activators on polar body emission following GVBD. TPA, 4 beta-PDD or diC8, but not 4 alpha-PDD or db-cAMP, inhibited polar body emission in a dose-dependent manner. The morphology and cytology of oocytes in which polar body emission was inhibited by TPA or 4 beta-PDD differed from that of oocytes treated with diC8. Thirty to 60% of the former were round in shape and exhibited a clump of chromosomes but no spindle; the remainder were distended in shape and exhibited a metaphase I spindle. All oocytes treated with diC8, however, were round, had dispersed chromosomes, and no spindle. These results suggest that, in contrast to resumption of meiosis, polar body emission is inhibited by activation of protein kinase C but not cAMP-dependent protein kinase.     

Cellular progesterone receptor phosphorylation in response to ligands activating protein kinases

Progesterone receptors were immunoprecipitated with monoclonal antibodies KD68 from lysates of human breast carcinoma T47D cells labelled to steady state specific activity with 32Pi. The 120 kDa 32P-labelled progesterone receptor band was resolved by polyacrylamide gel electrophoresis and identified by autoradiography. Phosphoamino acid analysis revealed serine phosphorylation, but no threonine or tyrosine phosphorylation. Treatment of the 32Pi-labelled cells with EGF, TPA or dibutyryl cAMP had no significant quantitative effect on progesterone receptor phosphorylation, though the EGF receptor and the cAMP-dependent protein kinases have been reported to catalyze phosphorylation of purified avian progesterone receptor preparations in cell free systems. Progesterone receptor phosphorylation on serine residues was increased by 2-fold in cells treated with 10 nM progesterone; EGF had no effect on progesterone-mediated progesterone receptor phosphorylation.     

In situ phosphorylation of human platelet myosin heavy and light chains by protein kinase C

Treatment of human platelets with 162 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in phosphorylation of a number of peptides, including myosin heavy chain and the 20-kDa myosin light chain. The site phosphorylated on the myosin heavy chain was localized by two-dimensional peptide mapping to a serine residue(s) in a single major tryptic phosphopeptide. This phosphopeptide co-migrated with a tryptic peptide that was produced following in vitro phosphorylation of platelet myosin heavy chain using protein kinase C. The sites phosphorylated in the 20-kDa myosin light chain in intact cells were analyzed by two-dimensional mapping of tryptic peptides and found to correspond to Ser1 and Ser2 in the turkey gizzard myosin light chain. In vitro phosphorylation of purified human platelet myosin by protein kinase C showed that in addition to Ser1 and Ser2, a third site corresponding to Thr9 in turkey gizzard myosin light chain is also phosphorylated. The phosphorylatable myosin light chains from human platelets were found to consist of two major isoforms present in approximately equal amounts, but differing in their molecular weights and isoelectric points. A third, minor isoform was also visualized by two-dimensional gel electrophoresis. Following treatment with TPA, both the mono- and diphosphorylated forms of each isoform could be visualized, and the sites of phosphorylation were identified. The phosphate content rose from negligible amounts found prior to treatment with TPA to 1.2 mol of phosphate/mol of myosin light chain and 0.7 mol of phosphate/mol of myosin heavy chain following treatment. These results suggest that TPA mediates phosphorylation of both myosin light and heavy chains in intact platelets by activation of protein kinase C.     

Des-Arg9 bradykinin modulates DNA synthesis, phospholipase C, and protein kinase C in cultured mesangial cells. Distinction from effects of bradykinin

The effects of the neuropeptide bradykinin (BK) and its natural proteolytic fragment Des-Arg9 bradykinin (DBK) on DNA synthesis and phospholipase C activation were investigated in cultured mesangial cells. DBK, acting through a distinct bradykinin receptor, induced DNA synthesis in serum-starved cultured mesangial cells. The effect of DBK was dose dependent (ED50 = 0.6 microM) and was strongly potentiated by insulin. Under the same conditions, BK had no effect. Down-regulation of protein kinase C by long term pretreatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) markedly reduced DBK-induced DNA synthesis. In the same way, co-incubation with the protein kinase C inhibitor staurosporine potently attenuated the response to DBK, suggesting a role of protein kinase C in DBK-induced mitogenesis. Analysis of phosphoproteins from 32P-labeled mesangial cells by two-dimensional gel electrophoresis revealed that DBK, like TPA but not BK, induced a net increase in the phosphorylation of an acidic cellular protein migrating with an apparent Mr = 80,000 (termed 80K), identified as a major and specific substrate of protein kinase C. Phosphorylation of the 80K protein by DBK or TPA was completely abolished in cells depleted of protein kinase C. DBK and TPA also induced an increase in phosphorylation of an Mr = 28,000 protein. Moreover, DBK but not TPA stimulated the phosphorylation of an Mr = 18,000 protein in normal as well as in protein kinase C-depleted cells. Analysis of phospholipase C activation revealed that DBK induced a large and sustained increase in diacylglycerol production and inositol phosphate accumulation over a 10-min incubation. BK had only a minor effect on both parameters. These results demonstrate that DBK, but not BK, modulates DNA synthesis through protein kinase C activation in cultured mesangial cells.     

Tumor promoter-dependent phosphorylation of a Triton X-100 extractable form of lipocortin I in T51B rat liver cells

The phosphorylation of lipocortin (a substrate of EGF-receptor kinase, and a putative phospholipase A2 inhibitor) was examined in T51B cells. By using Western blot procedures and antisera specific to lipocortin I, we found that most immunoreactive lipocortin I was located in the cytosol (lipocortin(cvt] of cells extracted in Ca2+-free buffers These cells however had another pool of immunoreactive lipocortin I located in the particulate fraction that was Triton X-100 extractable (lipocortin(mem]. Increasing Ca2+ concentrations in the extraction buffer resulted in more lipocortin(mem) recovered. In vitro phosphorylation of endogenous proteins demonstrated that lipocortin I became phosphorylated in a Ca2+ and phosphatidylserine-dependent manner, suggesting an involvement of protein kinase C. Treatment of cells with 100 ng/ml 12-0-tetradecanoylphorbol-13-acetate (TPA) but not with 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD) resulted in the in vitro phosphorylation of lipocortin(mem) by protein kinase C. TPA also increased the phosphorylation of lipocortin(mem) in [32P]phosphate-labeled cells.     

Effect of an exogenous auxin on maize tissues. Alteration of protein synthesis and phosphorylation

Pérez, L., Aguilar, R. and Sánchez-de-Jiménez, E. 1987. Effect of an exogenous auxin on maize tissues. Alteration of protein synthesis and phosphorylation. - Physiol. Plantarum 69: 517–522.
A synthetic auxin 2-(2-methyl-4-chloro)phenoxypropionic acid (MCPP), analogue of 2,4-D, alters maize ( Zea mays L. H-30) germination while inducing callus formation. The effect of this auxin on protein synthesis and phosphorylation of the embryonic tissues was explored. Total cytoplasmic proteins were analysed for ¹⁴C or ³²P incorporation into trichloroacetic acid precipitable material. MCPP significantly stimulated protein synthesis as well as protein phosphorylation. The protein synthesis pattern was highly altered in the presence of MCPP as analysed by two-dimensional gel electrophoresis. Analyses by Sephadex G-100 chromatography and by two-dimensional gel electrophoresis of phosphorylated proteins indicate that the effect of MCPP on protein phosphorylation was only quantitative.     

Dephosphorylation of the retinoblastoma protein during differentiation of HL60 cells.

Immunoprecipitated retinoblastoma protein from HL60 cells migrated as a series of bands during electrophoresis. The heterogeneity appeared to be generated by phosphorylation of the retinoblastoma protein. Treatment of the cells with the phorbol ester, tetradecanoyl phorbol acetate (TPA), resulted in both a loss of the heterogeneity of the pRB species and a significant decrease in the level of pRB phosphorylation. These changes accompanied differentiation of the HL60 cells into macrophages. Treatment of the cells with dibutyryl cAMP also resulted in dephosphorylation of pRB as well as cell cycle arrest, although no recognizable differentiation occurred. These results are consistent with a model in which TPA and dibutyryl cAMP dependent pathways can activate pRB by altering its phosphorylation.     

Phorbol diester-induced phosphorylation of nuclear matrix proteins in HL60 promyelocytes. Possible role in differentiation studied by cationic detergent gel electrophoresis

Immortal HL60 promyelocytes are induced to differentiate to mortal adherent cells by a variety of agents which activate protein kinase C, including 12-O-tetradecanoylphorbol 13-acetate (TPA). In order to investigate the mechanism of this effect, we incubated HL60 cells with [32P]orthophosphate with or without TPA and extracted their proteins with the cationic detergent benzyldimethyl-n-hexadecylammonium chloride prior to electrophoresis in a discontinuous polyacrylamide gel system in the first dimension. In this system, proteins migrate toward the cathode as a function of their molecular weight, and they are separated from other radioactive components which can obscure the pattern of protein phosphorylation on sodium dodecyl sulfate (SDS) gels. SDS gel electrophoresis was used in the second dimension, resulting in the clear resolution of a large number of proteins. TPA caused many changes in the pattern of protein phosphorylation in intact cells. Two proteins which prominently increased their incorporation of 32P were investigated in particular, and they were both found to be retained in the nuclear matrix following successive extraction of cells with Triton, digestion with DNase and RNase, and extraction with 2 M NaCl. These proteins migrated with apparent molecular weights of 80,000 and 33,000 on SDS gels, and are designated NP80 and NP33, respectively. NP80 was half-maximally phosphorylated after 7 min exposure to TPA, and half-maximally phosphorylated by 10 nM TPA. NP80 co-migrated with a faint Coomassie Blue-stained protein, and NP33 co-migrated with a more prominent protein. Several proteins incorporated less 32P when the cells were exposed to TPA, including one which was extracted from nuclei with the core histones and which co-migrated with histone H2A. Further study will be needed to determine whether the differentiation of HL60 induced by TPA is mediated via phosphorylation of these nuclear matrix proteins.     

Protein phosphorylation and secretion in digitonin-permeabilized adrenal chromaffin cells. Effects of micromolar Ca2+, phorbol esters, and diacylglycerol

The effects of phorbol esters, dioctanoylglycerol (DiC8), and micromolar Ca2+ on protein phosphorylation and catecholamine secretion in digitonin-treated chromaffin cells were investigated. [gamma-32P]ATP was used as a substrate for phosphorylation in the permeabilized cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) enhanced Ca2+-dependent catecholamine secretion from digitonin-permeabilized cells. The enhancement required MgATP. Only those phorbol esters which activate protein kinase C in vitro enhanced both catecholamine secretion and protein phosphorylation. DiC8, which activates protein kinase C in vitro and mimics phorbol ester effects in situ, also enhanced both catecholamine secretion and protein phosphorylation. Preincubation of intact cells with TPA or DiC8 was necessary for maximal effects on both catecholamine secretion and protein phosphorylation in subsequently digitonin-treated chromaffin cells. The TPA-induced enhancement of protein phosphorylation was almost entirely Ca2+-independent, whereas DiC8-induced enhancement of protein phosphorylation was mainly Ca2+-dependent. Micromolar Ca2+ alone also enhanced the phosphorylation of a large number of proteins. Most of the proteins phosphorylated in response to TPA or potentiated by DiC8 in combination with Ca2+ were also phosphorylated by micromolar Ca2+ in the absence of exogenous protein kinase C activators. In intact cells, 1,1-dimethyl-4-phenylpiperazinium (DMPP) induced Ca2+-dependent phosphorylation of at least 17 proteins which were detected by two-dimensional gel electrophoresis. All of the proteins phosphorylated upon incubation with 1,1-dimethyl-4-phenylpiperazinium were phosphorylated upon incubation with micromolar Ca2+ in digitonin-treated cells. These results demonstrate that TPA- or DiC8-enhanced Ca2+-dependent catecholamine secretion is associated with enhanced protein phosphorylation which is probably mediated by protein kinase C and that activation of protein kinase C modulates catecholamine secretion from digitonin-treated chromaffin cells.     

Lymphoma protein kinase C is associated with the transmembrane glycoprotein, GP85, and may function in GP85-ankyrin binding

In this study, several complementary techniques have been used to investigate the involvement of a protein kinase C (PKC) molecule in the plasma membrane-cytoskeleton interactions that occur in mouse T-lymphoma cells. Our data indicate that the lymphoma plasma membrane contains a 78-kDa polypeptide that exists in a complex with one of the major transmembrane glycoproteins, GP85 (a wheat germ agglutinin-binding protein). This membrane-associated 78-kDa protein appears to have PKC-like properties based on the following criteria: 1) it cross-reacts with a specific antibody raised against brain PKC; 2) it has a pI of 5.6-5.8, which is similar to that of the PKC described previously in other cell types; and 3) it displays characteristic PKC enzymatic activity by phosphorylating histone H1 in a Ca2+- and phospholipid-dependent manner. Double immunocytochemical staining experiments reveal that the lymphoma PKC-like molecules translocate from the cytoplasm to the cell membrane and accumulate directly underneath receptor capped structures following addition of various ligands. Studies we have done to identify the cellular substrate(s) of the lymphoma plasma membrane-associated PKC have shown that GP85 is preferentially phosphorylated in isolated membrane preparations following addition of the PKC activator, TPA (phorbol-12-O-tetradecanoyl-phorbol 13-acetate), but not the biologically inactive TPA analogue, 4 alpha-PDD (4 alpha-phorbol 12,13-didecanoate). In addition, we have found that GP85 can be phosphorylated by purified brain protein kinase C. Analysis of the resulting phosphoamino acids indicates that phosphorylation of GP85 occurs primarily at serine residues, occurs in minor amounts (approximately 5%) at threonine residues, and does not occur at tyrosine residues. These data indicate that the lymphoma GP85 is a substrate for PKC. Furthermore, we have established that phosphorylation of GP85 by PKC enhances its binding affinity with the membrane linker molecule, ankyrin. These findings suggest that PKC-mediated phosphorylation of GP85 may be an important part of the lymphoma plasma membrane-cytoskeleton interaction.     

Phagocytosis induced by thyrotropin in cultured thyroid cells is associated with myosin light chain dephosphorylation and stress fiber disruption

The actin/myosin II cytoskeleton and its role in phagocytosis were examined in primary cultures of dog thyroid cells. Two (19 and 21 kD) phosphorylated light chains of myosin (P-MLC) were identified by two- dimensional gel electrophoresis of antimyosin immunoprecipitates, and were associated with the Triton X-100 insoluble, F-actin cytoskeletal fraction. Analyses of Triton-insoluble and soluble 32PO4-prelabeled protein fractions indicated that TSH (via cAMP) or TPA treatment of intact cells decreases the MLC phosphorylation state. Phosphoamino acid and tryptic peptide analyses of 32P-MLCs from basal cells showed phosphorylation primarily at threonine and serine residues; most of the [32P] appeared associated with a peptide containing sites typically phosphorylated by MLC kinase. Even in the presence of the agents which induced dephosphorylation, the phosphatase inhibitor, calyculin A, caused a severalfold increase in MLC phosphorylation at several distinct serine and threonine sites which was also associated with actomyosin and cell contraction. Phosphorylation of cell homogenate proteins or the cytoskeletal fraction with [gamma-32P]ATP indicated that Ca2+, EGTA, or trifluoperazine (TFP) has little effect on the phosphorylation of MLC. Both fluorescent phalloidin and antimyosin staining of cells showed distinct dorsal and ventral stress fiber complexes which were disrupted within 30 min by TSH and cAMP; TPA appeared to cause disruption of dorsal, and rearrangement of ventral complexes. Concomitant with MLC dephosphorylation and stress fiber disruption, TSH/cAMP, but not TPA, induced dorsal phagocytosis of latex beads. While stimulation of either A or C-kinase disrupts dorsal stress fibers and rearranges actomyosin, another event(s) mediated by A-kinase appears necessary for phagocytic activity.     

The intermediate filament-lamina-nuclear matrix system of BHK-21 cells

We have employed collodial gold immuno-labelling in whole-mount cell and 2-D gel electrophoresis to demonstrate the intermediate filament (IF)-lamina-nuclear matrix (NM) system in BHK-21 (Baby Hamster Kidney) cells. Grown on grids, cells were gently extracted with salt solutions as previously described by S. Penman to preserve intact IF-lamina-NM systems. The extracted samples were fixed, postfixed, dehydrated and dried through the CO2 critical point, then examined under high voltage electron microscope (HVEM). The results revealed that the IF-lamina-NM system is a interconnecting network throughout the cell from cytoplasma to nuclear. The IF unit is 10 nm in diameter. IFs radiate away from the nuclear region into the spreading cytoplasm and the polarity of their distributing is obvious. The IF system closely connected to lamina. Immuno-gold labelling and 2-D gel proved that vimentin, a 55 KD protein (pI 5,6), is the major component of IFs in BHK-21 cells. Lamina can be precisely and specifically labelled with anti-lamin A, C proteins and as well as 2-D gel electrophoresis indicated that there are lamin A, B, C proteins in BHK-21 cells, whose molecular weights are 68 KD, 70 KD, 62 KD respectively. Its components are more complicated, but a few dots of NM proteins can be clearly distinguished in 2-D gel map, in which actin, a 45 KD protein (pI 4.5), might be involved. The nuclear matrix network was also clearly presented under HVEM. Its filaments can be labelled with anti-NM 298 KD protein precisely.     

BHK_(21)细胞的中间纤维-lamina-核骨架体系

以系列选择性抽提技术与显示细胞骨架的整装电镜技术为基础,应用免疫胶体金标记与蛋白质成份的双向电泳分析技术,研究了BHK_(21)细胞的中间纤维-lamina与核骨架(核基质)结构体系及其主要的蛋白成份。BHK_(21)细胞的中间纤维-lamina与核骨架是在结构上相互联系,贯穿于核与质的网络体系。中间纤维单丝直径为10nm,能很好地被抗波形蛋白抗体-金颗粒所标记,生化分析同样说明BHK_(21)细胞中间纤维的主要成份是波形蛋白(vimentin),其分子量为55KD,等电点为5.6。中间纤维网在胞质内呈极性分布,与lamina密切联结。BHK_(21)细胞的lamina能被抗lamin A与C的单克隆抗体-金颗粒标记。双向电泳分析证明,lamina含有三种蛋白成份,即lamin A,B,C,其分子最分别为68KD,70KD与62KD,lamin A,C等电点均为6.9—7.2,而lamin B偏酸,其等电点为5.8。BHK_(21)细胞核骨架纤维网也可以被清晰的显示,其蛋白成份较为复杂,在双向电泳谱上经常出现多个清晰的斑点,很可能含有肌动蛋白(actin)。298KD核基质蛋白的单克隆抗体-金颗粒能准确的标记核骨架纤维。     

Temporal patterns of protein phosphorylation after angiotensin II, A23187 and/or 12-O-tetradecanoylphorbol 13-acetate in adrenal glomerulosa cells.

The temporal patterns of protein phosphorylation in the adrenal glomerulosa cell were analysed by two-dimensional electrophoresis after stimulation with 10 nM-angiotensin II or various agents [10 nM-12-O-tetradecanoylphorbol 13-acetate (TPA), 50 nM-A23187, 1 microM-nitrendipine], administered singly or in combination. These patterns were compared with the temporal patterns of aldosterone secretion induced by the same agonists and antagonists. After 1 and 30 min of stimulation with angiotensin II, different patterns of protein phosphorylation were observed. A comparison of these patterns reveals that: the phosphorylation of only one protein was persistently enhanced during the continuous incubation with angiotensin II; the phosphorylation of five proteins was transiently enhanced (at 1 min but not 30 min); and the phosphorylation of three proteins did not occur at 1 min but was seen at 30 min. Addition of the phorbol ester TPA alone, which at 30 min is without effect in enhancing aldosterone production, has no effect on protein phosphorylation. The combined addition of TPA and the Ca2+ ionophore, A23187, which, like angiotensin II, evokes a sustained increase in aldosterone production, reproduced the temporal patterns of protein phosphorylation seen after angiotensin II action. Manipulations (A23187 alone, angiotensin II plus nitrendipine) which evoke only a transient rise in aldosterone production rate induce a transient rise in cellular protein phosphorylation. The 1 min patterns of phosphorylation seen after A23187 or combined angiotensin II and nitrendipine (a Ca2+ channel antagonist) are similar to those observed after 1 min of angiotensin II stimulation. These results suggest that, when angiotensin II acts, the initial cellular response is mediated by a different mechanism than that responsible for the sustained response.     

Phosphorylation of recombinant non-NMDA glutamate receptors on serine and tyrosine residues

Glutamate receptors are the major excitatory neurotransmitter receptors in the central nervous system. A variety of data has recently suggested that protein phosphorylation of glutamate receptors regulates their function. To examine at a molecular level the role of protein phosphorylation in the modification of glutamate receptors, we have transiently expressed the non-NMDA glutamate receptor subunit GluR1 (flop) in human embryonic kidney 293 cells. Using a polyclonal antipeptide antiserum directed specifically against GluR1, we have immunoprecipitated a 106 kDa phosphoprotein corresponding to the GluR1 subunit. Phosphoamino acid analysis and thermolytic peptide mapping demonstrate that this basal phosphorylation occurs exclusively on serine residues in two phosphopeptides. Application of activators of endogenous cAMP-dependent protein kinase or protein kinase C revealed no consistant changes in the phosphorylation of GluR1. However, coexpression of the GluR1 subunit with the well characterized protein tyrosine kinase v-src results in phosphorylation of GluR1 on tyrosine residues, in a single thermolytic phosphopeptide. These results suggest that GluR1 may be a substrate for protein serine/threonine kinases as well as protein tyrosine kinases in the central nervous system.Abbreviations AMPA -amino-3-hydroxy-5-methyl-4-isoxazolepropionate - CNS central nervous system - NMDA N-methyl-D-aspartate; - PAGE polyacrylamide gel electrophoresis - PBS phosphate-buffered saline - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate - TBS Tris-buffered saline - TPA phorbol 12-myristate-13-acetate Special issue dedicated to Dr. Paul Greengard.     

Changes in the phosphorylation status of the 27 kDa heat shock protein (HSP27) associated with the modulation of growth and/or differentiation in MCF-7 cells

We have used human mammary cells of the MCF-7 strain, which constitutively express high levels of the small heat shock protein HSP27 and we have compared the changes in the phosphorylation status of this protein together with changes in cell growth and/or morphology induced by the action of one of the following agents: (1) TPA (12-O-tetradecanoylphorbol-13-acetate), known as a differentiation inducer in MCF-7 cells; (2) OH-TAM (hydroxytamoxifen), which exerts a cytostatic and cytotoxic action; or (3) TNFα (tumour necrosis factor), which induces apoptotic cell death in this cell line. Our data show that TPA and TNF stimulate an immediate and massive phosphorylation of HSP27, whereas OH-TAM affect the phosphorylation status of the protein only after a 3 day delay. In the case of TPA, high levels of HSP27 phosphorylation were maintained for at least 4 days, along with growth inhibition and acquisition by the cells of a secretory phenotype. TPA and OH-TAM exerted similar immediated effects on cell growth, despite the different time course of their action on HSP27 phosphorylation. This excludes the possibility that the latter is a necessary consequence of, or an absolute requisite to, growth inhibition. With OH-TAM and TNF the increase in HSP27 phosphorylation was concomitant with the appearance of apoptosis, not observed with TPA. This indicates that increased phosphorylation of HSP27 is not specifically associated with the triggering or the execution of apoptosis in these cells. Altogether, our data support the concept that phosphorylated HSP27 is involved (and might then be rate limiting in some instances) in the execution of vital cell programmes (including resistance to stress, proliferation and differentiation), as well as in that of cell death. This is consistent with its role in actin polymerization and its position downstream of the p38/RK-type MAPkinase, itself a point of convergence for diverse signal transduction pathways.     

Interactions between cyclic AMP- and phorbol ester-dependent phosphorylation systems in S49 mouse lymphoma cells

High-resolution two-dimensional gel electrophoresis of proteins labeled with either 32Pi or [35S]methionine was used to study interactions between cyclic AMP and tetradecanoyl phorbol acetate (TPA) at the level of intracellular protein phosphorylation. Cultured S49 mouse lymphoma cells were used as a model system, and mutant sublines lacking either the catalytic subunit of cyclic AMP-dependent protein kinase or the guanyl nucleotide-binding "Ns" factor of adenylate cyclase provided tools to probe mechanisms underlying the interactions observed. Three sets of phosphoproteins responded differently to TPA treatment of wild-type and mutant cells: Phosphorylations shown previously to be responsive to activation of intracellular cyclic AMP-dependent protein kinase were stimulated by TPA in wild-type cells but not in mutant cells, a subset of phosphorylations stimulated strongly by TPA in mutant cells was inhibited in wild-type cells, and two novel phosphoprotein species appeared in response to TPA only in wild-type cells. The latter two classes of TPA-mediated responses specific to wild-type cells could be evoked in adenylate cyclase-deficient cells by treating concomitantly with TPA and either forskolin or an analog of cyclic AMP. Three conclusions are drawn from our results: 1) TPA stimulates adenylate cyclase in wild-type cells causing increased phosphorylation of endogenous substrates by cyclic AMP-dependent protein kinase, 2) activated cyclic AMP-dependent protein kinase inhibits phosphorylation (or enhances dephosphorylation) of a specific subset of TPA-dependent phosphoproteins, and 3) cyclic AMP-dependent events facilitate TPA-dependent phosphorylation of some substrate proteins.     

Purification and characterization of a calcium-unresponsive, phorbol ester/phospholipid-activated protein kinase from porcine spleen

A calcium-unresponsive, phorbol ester/phospholipid-activated protein kinase was purified to apparent homogeneity from a Triton X-100 extract of an EGTA/EDTA-preextracted particulate fraction of porcine spleen by chromatography on S-Sepharose Fast Flow, phenyl-Sepharose Fast Flow, protamine-agarose, and Superdex 200. The enzyme had a Mr of 76,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (p76-kinase). A similar value (78,000) was obtained by gel filtration. The purified p76-kinase proved to be much more stable than the enzyme in crude preparations. Storage in a buffer containing 50 mM mercaptoethanol and 20% glycerol at -20 degrees C for at least 4 months caused less than 20% loss in enzyme activity. The enzyme exhibited a pH optimum of 8.3. The affinity of the novel enzyme for substrates and cofactors differed to some extent from that of conventional alpha, beta, gamma protein kinase C (PKC). p76-kinase did not respond to calcium, had a lower requirement for magnesium, and a higher affinity for histone III-S than PKC. Both the p76-kinase-catalyzed phosphorylation of histone III-S and the autophosphorylation of the enzyme could be activated by the phorbol ester TPA (or diacylglycerol) plus phosphatidyl serine, but not by calcium plus phosphatidyl serine. The stoichiometry of autophosphorylation suggested that fully phosphorylated p76-kinase contained two phosphoserine residues and one phosphothreonine residue. Like PKC, p76-kinase bound TPA with high affinity (KD = 9.6 nM). In the absence of TPA, various unsaturated fatty acids, particularly arachidonic acid, were more potent as activators of the enzyme than phosphatidyl serine. The p76-kinase was recognized by an antiserum raised against a delta PKC-specific peptide, but not by an alpha, beta, gamma PKC-specific antiserum. The previously described p82-kinase of mouse epidermis and spleen exhibiting the same properties as the p76-kinase did also react with the p76-kinase-specific antiserum.     

Phosphorylation of class I histocompatibility antigens in human B lymphocytes. Regulation by phorbol esters and insulin.

Phosphorylation of membrane proteins is one of the earliest steps in cell activation induced by growth-promoting agents. Since MHC (major histocompatibility complex) class I molecules are known to contain phosphorylation sites in their C-terminal intracellular domain, we have studied the regulation of HLA (human leucocyte antigen) phosphorylation in intact cells by two mitogens, namely TPA (12-O-tetradecanoylphorbol 13-acetate), a phorbol ester, and insulin, which are thought to exert their mitogenic effects through the stimulation of different protein kinases (protein kinase C and a tyrosine kinase respectively). Human B lymphoblastoid cells (526 cell line) were pulsed with [32P]Pi to label the intracellular ATP pool. Cells were then stimulated for 10 min with TPA, insulin, cyclic AMP or EGF (epidermal growth factor). The reaction was stopped by cell lysis in the presence of kinase and phosphatase inhibitors, and class I HLA antigens were immunoprecipitated with monoclonal antibodies. Analysis of labelled proteins by gel electrophoresis and autoradiography revealed that TPA increased the phosphorylation of the 45 kDa class I heavy chain by 5-7-fold, and insulin increased it by 2-3-fold. Cyclic AMP and EGF had no stimulatory effect. Analysis of immunoprecipitated HLA molecules by two-dimensional gel electrophoresis showed that TPA and insulin stimulated the incorporation of 32P into different 45 kDa molecular species, suggesting that different sites were phosphorylated by two agents. Moreover, incubation of purified class I MHC antigens with partially purified insulin-receptor tyrosine kinase and [gamma-32P]ATP revealed that class I antigens could also be phosphorylated in vitro by this tyrosine kinase. Altogether, these results therefore confirm that insulin receptors and HLA class I molecules are not only structurally [Fehlmann, Peyron, Samson, Van Obberghen, Brandenburg & Brossette (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 8634-8637] but also functionally associated in the membranes of intact cells.