Protein phosphorylation in response to stress in Clostridium acetobutylicum

The possible involvement of protein phosphorylation in the clostridial stress response was investigated by radioactively labeling growing cells of Clostridium acetobutylicum with 32Pi or cell extracts with [gamma-32P]ATP. Several phosphoproteins were identified; these were not affected by the growth stage of the culture. Although the extent of protein phosphorylation was increased by heat stress, the phosphoproteins did not correspond to known stress proteins seen in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified clostridial DnaK, a stress protein, acted as a kinase catalyzing the phosphorylation of a 50-kilodalton protein. The phosphorylation of this protein was enhanced in extracts prepared from heat-stressed cells. Diadenosine-5',5"'-P1,P4-tetraphosphate had no influence on protein phosphorylation.     

Extracellular phosphorylation in the parasite, Leishmania major

Intact promastigotes or cell-free extracts of the parasite Leishmania major were labelled with adenosine 5'[gamma-32P]-triphosphate (ATP). This resulted in the identification of eleven phosphoproteins. [gamma-32P]ATP incorporation into endogenous and exogenous substrates was insensitive to most of the commonly used protein kinase inhibitors and activators indicating that the leishmanial enzyme(s) may represent a new class of kinase(s). In addition, exogenous substrate specificity was inconsistent with the preferences of second messenger-dependent protein kinases. Cyclic AMP had differential effects on phosphorylation in intact cells and lysates. The majority of kinase activity could be attributed to an externally oriented membrane-associated protein kinase(s), as no specific cytosolic phosphoproteins were found and intact cells phosphorylated exogenous substrates. Labelled ATP did not cross the membrane and [alpha-32P]ATP was an unsuitable substrate for the phosphorylation activity. The ectokinase activity on live Leishmania exhibited a different substrate preference when compared to the protein kinase activity in the particulate fraction, suggesting that more than one protein kinase may be present in L. major. Three serine-labelled phosphoproteins were specifically released into the medium. The presence of an ecto-kinase and these released phosphoproteins may play a significant role in host-parasite interactions.     

Evidence of protein-tyrosine kinase activity in the bacterium Acinetobacter calcoaceticus

Protein phosphorylation was investigated in the bacterium Acinetobacter calcoaceticus both in vivo and in vitro. In cells grown with [32P]orthophosphate, several radioactive phosphoproteins were detected by gel electrophoresis and autoradiography. These proteins were shown to contain phosphoserine, phosphothreonine, and a relatively large proportion of phosphotyrosine residues. Incubation of cellular extracts with [gamma-32P] ATP also resulted in the phosphorylation of several proteins. At least four of them, namely an 81-kDa protein, were modified at tyrosine. No protein labeling occurred when extracts were incubated with [gamma-32P] ATP or [14C]ATP. Moreover, phosphoproteins were insensitive to snake venom phosphodiesterase. All together these results indicate that A. calcoaceticus harbors different protein kinases including a protein-tyrosine kinase activity. Further analysis of this activity showed that it has little, if any, functional similarity with eukaryotic protein-tyrosine kinases.     

Protein phosphorylation in normal and neoplastic development. Phosphorylation of proteins endogenous to foetal tissues and tumours.

The abilities of proteins endogenous to normal and neoplastic tissues to serve as substrates in a protein-phosphorylation reaction in vitro were compared. After the tissue extracts were incubated with [gamma-32P]ATP, the phosphorylated proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the dried gels were subjected to radioautography. Considerable incorporation of 32P into a protein of mol.wt. 135000 was observed with extracts from foetal tissues and tumours, but only minimal incorporation into this protein occurred when extracts from adult tissues were used. The ability of this protein to become phosphorylated in vitro may be related to cell proliferation. When ascites cells were incubated with [32P]Pi, one of the major phosphoproteins migrated on sodium dodecyl suphate/polyacrylamide gels at mol.wt. 135000, suggesting that this protein can be phosphorylated both in intact cells and broken-cell preparations. A protein of mol.wt. 87000 was highly phosphorylatable in extracts from solid tumours, but was not phosphorylated in extracts from ascites tumours, foetal or adult tissues. The phosphorylation pattern of these two proteins can thus distinguish solid neoplasms and normal adult tissues from ascites tumours and from foetal tissues. A protein of mol.wt. 49000, which was the most labelled protein in adult tissues, was also one of the major phosphoproteins in foetal and neoplastic tissues. Numerous mechanisms are postulated to explain how the extent of 32P incorporation into a protein could vary as a function of biological state.     

Protein phosphorylation in rat mammary acini and in cytosol preparations in vitro. Phosphorylation of acetyl-CoA carboxylase is unaffected by cyclic AMP.

Phosphorylation of soluble proteins in rat mammary acinar cells was investigated. When phosphorylation proceeded in intact cells, in the presence of [32P]Pi, the major non-casein phosphoproteins, including acetyl-CoA carboxylase, were unresponsive to incubation conditions that caused major increases in the intracellular concentration of cyclic AMP. The overall 32P specific radioactivity (c.p.m./microgram of protein) of acetyl-CoA carboxylase, assessed after affinity purification of the enzyme with avidin-Sepharose, was unchanged by incubation under such conditions. Furthermore, the distribution of 32P among tryptic phosphopeptides of the enzyme, resolved by reversed-phase h.p.l.c., was not altered by cyclic AMP-increasing treatments of the acinar cells. When cytosol fractions were incubated with [gamma-32P]ATP, some phosphoproteins responded to the addition of micromolar concentrations of dibutyryl cyclic AMP or cyclic AMP by undergoing an enhancement of phosphate incorporation. In these experiments in vitro, protein phosphatase activity did not make a major contribution to the net phosphorylation of individual phosphoproteins, and acetyl-CoA carboxylase was not prominent among the phosphoproteins identified after short (less than 1 min) incubations of cytosols with [gamma-32P]ATP. The resistance of protein phosphorylation to variations in the cyclic AMP concentration in intact mammary epithelial cells, demonstrated by this work, is one of several mechanisms that ensure the pleiotropic refractoriness of those cells to agents which normally cause a stimulation of adenylate cyclase activity in hormone-sensitive cells.     

Cell adhesion-dependent differences in endogenous protein phosphorylation on the surface of various cell lines

Endogenous phosphorylation of intact cells was studied with four mouse, hamster and human cell lines using [gamma-32P]ATP and [gamma-32P]GTP as exogenous substrates. With all four cell lines distinct differences in the phosphoprotein patterns could be demonstrated for cells grown in suspension culture compared to cells grown in monolayers. Two major, apparently ubiquitous phosphoproteins with molecular weights of 135 000 (128 000 in HeLa cells) and 105 000, representing up to 60% of total phosphorylation, were phosphorylated only in cells grown in suspension. These phosphoproteins and the kinase(s) were located on the surface of the suspension cells. Evidence showed that phosphorylation was apparently not a true endogenous reaction, that rather it occurred by cell-cell collision, showing exponentially increasing 32P incorporation with increasing cell population density. Phosphorylation of pp135 and pp105 was established with ATP as well as with GTP and was not dependent on cyclic nucleotides cyclic AMP, cyclic GMP and cyclic CMP. The substrate-attached cells of all four cell lines have protein kinases on the cell surface. The lack of pp135 and pp105 phosphorylation may be due to the fact that these phosphoproteins are not expressed at all on the surface of substrate-attached cells or that these phosphoproteins are already fully phosphorylated.     

Phosphoproteins and the phosphoenolpyruvate:sugar phosphotransferase system of Streptococcus salivarius. Detection of two different ATP-dependent phosphorylations of the phosphocarrier protein HPr

Phosphoproteins which arise from incubation of Streptococcus salivarius ATCC25975 crude extracts with [32P]phosphoenolpyruvate and [gamma-32P]ATP, were separated and detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. These procedures were carried out using the methodology that has been developed to allow for the detection of phosphoproteins containing 1-P-histidinyl and 3-P-histidinyl residues, and also to distinguish between these and phosphoproteins containing acid-stable phosphoamino acids such as phosphoserine, phosphothreonine, and phosphotyrosine. Extracts of cells which had been grown with various sugars as carbon sources were investigated to determine both constitutive and inducible phosphoproteins. No evidence was found for phosphoproteins specifically induced by a sugar, and in particular no evidence was found for any IIIsugar phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Incubation with [gamma-32P]ATP showed that histidine-containing phosphocarrier protein (HPr) of the PTS could be phosphorylated to give both acid-stable and acid-labile phosphoamino acid residues. The acid-labile ATP-dependent phosphorylation activity was activated by glucose-6-P and appeared to produce a 3-P-histidinyl residue in HPr.     

Serine phosphorylation of the v-rel oncogene product/pp40 complex

The transforming protein encoded by the v-rel oncogene of reticuloendotheliosis virus has been purified from REV-T transformed lymphoid cells by sequential DEAE-Sepharose and immunoaffinity chromatography. The purified preparation consisted of pp59v-rel and the 40 kDa cellular protein which is complexed with the v-rel oncogene product in transformed cells as well as some minor proteins. Incubation of this purified preparation in the presence of Mg2+ and (gamma-32P)ATP resulted in phosphorylation of both pp59v-rel and the 40 kDa protein. This preparation was also able to phosphorylate casein on serine residues. Immunoprecipitates obtained from extracts of REV-T transformed lymphoid cells labeled with 32P-orthophosphate contained 59 and 40 kDa phosphoproteins. Both pp59v-rel and the 40 kDa protein were phosphorylated on serine residues in transformed cells.     

Protein Phosphorylation in Astrocytes Mediated by Protein Kinase C: Comparison with Phosphorylation by Cyclic AMP-Dependent Protein Kinase

The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), has been found recently to transform cultured astrocytes from flat, polygonal cells into stellate-shaped, process-bearing cells. Studies were conducted to determine the effect of PMA on protein phosphorylation in astrocytes and to compare this pattern of phosphorylation with that elicited by dibutyryl cyclic AMP (dbcAMP), an activator of the cyclic AMP-dependent protein kinase which also affects astrocyte morphology. Exposure to PMA increased the amount of 32P incorporation into several phosphoproteins, including two cytosolic proteins with molecular weights of 30,000 (pI 5.5 and 5.7), an acidic 80,000 molecular weight protein (pI 4.5) present in both the cytosolic and membrane fractions, and two cytoskeletal proteins with molecular weights of 60,000 (pI 5.3) and 55,000 (pI 5.6), identified as vimentin and glial fibrillary acidic protein, respectively. Effects of PMA on protein phosphorylation were not observed in cells depleted of protein kinase C. In contrast to the effect observed with PMA, treatment with dbcAMP decreased the amount of 32P incorporation into the 80,000 protein. Like PMA, treatment with dbcAMP increased the 32P incorporation into the proteins with molecular weights of 60,000, 55,000 and 30,000, although the magnitude of this effect was different. The effect of dbcAMP on protein phosphorylation was still observed in cells depleted of protein kinase C. The results suggest that PMA, via the activation of protein kinase C, can alter the phosphorylation of a number of proteins in astrocytes, and some of these same phosphoproteins are also phosphorylated by the cyclic AMP-dependent mechanisms.     

Modulation in protein phosphorylation during G2 phase of the cell cycle in two-cell mouse embryos

The protein phosphorylation activities in extracts were assayed for 2-cell mouse embryos at three stages of the G2 phase of the cell cycle. The 2-cell embryos were unique in having a prolonged G2 phase and so easily staged at early G2 (EG2), middle G2 (MG2) and late G2 (LG2) by timing the embryo isolation from pregnant mice. The embryo extracts were used both as sources of protein kinases and their substrates. The phosphoproteins of the extracts were labelled with [gamma-32P]ATP and separated by electrophoresis on SDS-polyacrylamide gels. The present study revealed that protein phosphorylation increased 3-6-fold during the progression of 2-cell embryos from EG2 to LG2 and the level of protein phosphorylation at any stages was greatly decreased by the presence of cAMP. Thus, the protein phosphorylation system of 2-cell mouse embryos seems to differ from those reported systems in mammals in its negative dependence on cAMP.     

Phosphorylation of membrane-associated proteins by phorbol esters in isolated bag cell neurons of Aplysia

Following brief synaptic stimulation, the bag cell neurons in the abdominal ganglion of Aplysia undergo a series of changes in electrophysiological and secretory properties that triggers egg laying behavior. Activation of protein kinase C appears to play an important role in these changes and, in particular, causes the unmasking of a new species of voltage-dependent calcium channel. We have now used isolated bag cell neurons maintained in cell culture to study changes in protein phosphorylation that are induced by exposure to an activator of protein kinase C. Primary cultures of bag cell neurons were labeled with 32P orthophosphate and then incubated with either tetradecanoyl phorbol 13-acetate (TPA), a potent activator of protein kinase C, or with an inactive phorbol ester. When protein extracts were separated with 2D electrophoresis approximately 100 phosphoproteins could be distinguished. Only four of these proteins, with molecular weights of 20, 32, 200, and 250 kD, underwent a reproducible increase in the extent of phosphorylation of at least twofold in response to TPA. TPA-induced changes in phosphate incorporation were blocked by pretreatment with the protein kinase C inhibitor H7. One of the TPA-regulated phosphoproteins was localized in a plasma membrane-containing fraction and was sensitive to trypsin treatment of intact cells, suggesting that it is a membrane protein with sites exposed to the extracellular medium. Two of the other TPA-regulated phosphoproteins may be associated with the inner face of the plasma membrane. Our results indicate that only a small number of proteins undergo a major change in phosphorylation state following the activation of protein kinase C in isolated bag cell neurons. One or more of these proteins may contribute to the unmasking of the calcium channels.     

Regulation of mouse preimplantation development: inhibitory effect of genistein, an inhibitor of tyrosine protein phosphorylation, on cleavage of one-cell embryos

We investigated the effects of genistein, an inhibitor of tyrosine protein phosphorylation, on mouse 1-cell embryos, since in response to mitogenic stimuli tyrosine protein phosphorylation in somatic cells is implicated in initiation of DNA synthesis. Genistein inhibits cleavage of 1-cell embryos in a concentration-dependent and reversible manner; biochanin A, which is a less potent inhibitor of tyrosine protein phosphorylation, is a less potent inhibitor of cell cleavage. Genistein does not inhibit [35S]methionine incorporation, but does inhibit [3H]thymidine incorporation. Consistent with genistein's ability to inhibit cleavage by inhibiting DNA synthesis is that the loss of genistein's ability to inhibit cleavage corresponds with exit of the 1-cell embryos from S phase. Genistein is likely to inhibit tyrosine protein phosphorylation in situ, since it reduces by 80% the relative amount of [32P]phosphotyrosine present in 1-cell embryos; genistein does not inhibit either [32P]orthophosphate uptake or incorporation. As anticipated, genistein has little effect on inhibiting changes in the pattern of phosphoprotein synthesis during the first cell cycle, since tyrosine protein phosphorylation constitutes a small percentage of total protein phosphorylation. Alkalai treatment of [32P]radiolabeled phosphoproteins transferred to Immobilon reveals a base-resistant set of phosphoproteins of Mr = 32,000 that displays cell-cycle changes in phosphorylation. Although these properties suggest that these phosphoproteins may be related to the p34cdc2 protein kinase, phosphoamino acid analysis of [32P]radiolabeled phosphoproteins reveals that they are not enriched for phosphotyrosine; the inactive for p34cdc2 protein kinase contains a high level of phosphotyrosine. Results of these experiments suggest that tyrosine protein phosphorylation in response to the fertilizing sperm may be involved in initiating DNA synthesis in the 1-cell embryo, as well as converting a meiotic cell cycle to a mitotic one.     

Spermine-enhanced protein phosphorylation in human placenta

Polyamines are known to have a role in cell proliferation, differentiation, and protein synthesis. During pregnancy, major changes in polyamine levels occur in maternal serum, amniotic fluid, and placental tissue. Polyamine-activated phosphorylation has recently been proposed as a mechanism by which polyamines may regulate metabolic processes in target tissues. Polyamine-activated protein phosphorylation has not been studied in placenta. Homogenate membrane and cytosol fractions from human placenta were subjected to an endogenous protein phosphorylation assay using [gamma-32P]ATP in the presence and absence of the polyamines, spermine and spermidine, and the diamine, putrescine. Protein phosphorylation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. When compared to basal levels, spermine (10(-3) M) significantly (P less than 0.001) stimulated 32P incorporation into phosphoproteins having molecular weights of 55,000 and 105,000. At this concentration spermidine and putrescine failed to stimulate phosphorylation. Half-maximal 32P incorporation was observed with 3.7 +/- 1.25 X 10(-4) M spermine. Polylysine enhanced the phosphorylation of phosphoproteins of the same molecular weight as those enhanced by spermine. Heparin and high Mg2+ inhibited spermine-induced phosphorylation. cAMP and Ca2+ did not stimulate phosphorylation of the spermine-dependent phosphoproteins. Spermine, however, acted as an antagonist for cAMP-dependent phosphorylation of a Mr 45,000 phosphoprotein.     

Metaphase protein phosphorylation in Xenopus laevis eggs.

Cytoplasmic extracts of metaphase (M-phase)-arrested Xenopus laevis eggs support nuclear envelope breakdown and chromosome condensation in vitro. Induction of nuclear breakdown is inhibited by AMPP(NH)P, a nonhydrolyzable ATP analog, but not by ATP or gamma-S-ATP, a hydrolyzable ATP analog, suggesting that protein phosphorylation may be required for M-phase nuclear events in vitro. By addition of [gamma-32P]ATP, we have identified in cytoplasmic extracts and in intact eggs at least six phosphoproteins that are present during M-phase but absent in G1/S-phase. These phosphoproteins also appear in response to partially purified preparations of maturation-promoting factor. A subset of these proteins are thiophosphorylated by gamma-S-ATP under conditions that promote nuclear envelope breakdown and chromosome condensation. Each of these proteins is phosphorylated on serine and threonine, and one, a 42-kilodalton protein, is also phosphorylated on tyrosine both in extracts and in intact eggs. These results indicate that activation of protein kinases accounts for at least part of the increased phosphorylation in M-phase and that both protein-serine-threonine kinases and protein-tyrosine kinases may play a role in controlling M-phase nuclear behavior.     

Phosphorylation of proteins in Clostridium thermohydrosulfuricum.

Cell extracts of the thermophile Clostridium thermohydrosulfuricum catalyzed the phosphorylation by [gamma-32P]ATP of several endogenous proteins with Mrs between 13,000 and 100,000. Serine and tyrosine were the main acceptors. Distinct substrate proteins were found in the soluble (e.g., proteins p66, p63, and p53 of Mrs 66,000, 63,000, and 53,000, respectively) and particulate (p76 and p30) fractions, both of which contained protein kinase and phosphatase activity. The soluble fraction suppressed the phosphorylation of particulate proteins and contained a protein kinase inhibitor. Phosphorylation of p53 was promoted by 10 microM fructose 1,6-bisphosphate or glucose 1,6-bisphosphate and suppressed by hexose monophosphates, whereas p30 and p13 were suppressed by 5 microM brain (but not spinach) calmodulin. Polyamines, including the "odd" polyamines characteristic of thermophiles, modulated the labeling of most of the phosphoproteins. Apart from p66, all the proteins labeled in vitro were also rapidly labeled in intact cells by 32Pi. Several proteins strongly labeled in vivo were labeled slowly or not at all in vitro.     

Effect of lutropin on phosphorylation of endogenous proteins in testis Leydig cells. Correlation with testosterone production

The effect of lutropin on phosphorylation of endogenous proteins in testis Leydig cells was investigated, by incubating purified Leydig cells with lutropin and [(32)P]P(i) followed by sodium dodecyl sulphate/polyacrylamide-slab gel electrophoresis of the [(32)P]phosphoproteins. The radioactivity of the proteins was quantified by densitometry of the radio-autograms obtained. The following results were obtained. 1. Lutropin increased the amount of (32)P incorporated into three proteins (A, B and C) with apparent mol.wts. of 14300, 57000 and 77600 respectively. 2. The increase in incorporation of (32)P into these proteins was detectable within 5min, reaching a maximum in approx. 20min. 3. The (32)P incorporated into protein B (but not proteins A and C) was significantly increased with 0.1 and 1.0ng of lutropin/ml. Incorporation of (32)P into all three proteins was significantly increased with 10ng of lutropin/ml, reaching a maximum with 100ng/ml. 4. Testosterone production was significantly increased with 1ng of lutropin/ml, and between 10 and 1000ng/ml the degree of stimulation of testosterone production and incorporation of (32)P into proteins A, B and C was similar. 5. Cyclic AMP production was significantly increased with 10ng of lutropin/ml and had not reached a maximum with 1000ng/ml. 6. In Leydig cells isolated from hypophysectomized rats 3h after injection of choriogonadotropin in vivo, phosphoproteins with the same molecular weights as proteins A, B and C were found. No further increases in incorporation of (32)P into these proteins were obtained when lutropin was added to the Leydig cells in vitro. 7. Dibutyryl cyclic AMP (but not follitropin or testosterone) also stimulated the incorporation of (32)P into proteins A, B and C in Leydig cells.     

Comprehensive phosphoproteome analysis in rice and identification of phosphoproteins responsive to different hormones/stresses

Phosphoproteins in rice were detected by in vitro protein phosphorylation followed by two-dimensional polyacrylamide gel electrophoresis. Forty-four phosphoproteins were detected on a 2D-gel after in vitro protein phosphorylation of the crude extract from rice leaf sheath. Among the phosphoproteins detected, 42 were identified through analysis by Q-TOF MS/MS and/or MALDI-TOF MS. The largest percentage of the identified phosphoproteins are involved in signaling (30%), while 18% are involved in metabolism. When rice seedlings were treated with various hormones and stresses, it was observed that the phosphorylation of 13 proteins was enhanced differentially by different hormone and stress treatments. Furthermore, when the hormone/stress regulated phosphoproteins are compared in rice leaf sheath, leaf blade and root, only cytoplasmic malate dehydrogenase was found to be phosphorylated in all the tissues. Results suggest that in the phosphorylation cascade of rice, glycolytic metabolism processes and Ca(2+)-signaling seem to be important targets in response to hormones and stresses. Furthermore, the direct visualization of phosphoproteins by (32)P-labeling and their mass spectrometric identification provides an accurate and reliable method of analyzing the rice phosphoproteome.     

Comparative analysis of phosphoprotein-enriched myocyte proteomes reveals widespread alterations during differentiation

The differentiation of skeletal muscle has been associated with altered phosphorylation status of individual proteins. However, a global analysis of protein phosphorylation during myogenesis has yet to be undertaken. Here, we report the identification of over 130 putative phosphoproteins from murine C2C12 muscle cells. Cell extracts were fractionated on phosphoprotein enrichment columns and the resulting proteins were detected by two-dimensional gel electrophoresis and silver stain, and identified by liquid chromatography coupled to electrospray tandem mass spectrometry. The early differentiation of C2C12 myoblasts was found to be accompanied by changes in the phosphorylation or expression of numerous proteins including cytoskeletal, heat shock and signaling proteins, the pp32 family of nuclear phosphoproteins, several disease-associated gene products and other characterized and uncharacterized proteins.     

Calcium-phospholipid enhanced protein phosphorylation in human placenta

Calcium-activated, phospholipid-dependent protein phosphorylation has not been studied in placenta. Human placental cytosol was subjected to an endogenous protein phosphorylation assay using [gamma-32P]ATP in the presence of calcium and phosphatidylserine. Protein phosphorylation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. When compared to basal levels, calcium (10(-6) M) in combination with phosphatidylserine (50 micrograms/ml) significantly enhanced (P less than 0.01) 32P incorporation into phosphoproteins having mol wt 47,000, 43,000, and 37,000. Half-maximal 32P incorporation was observed with 3.5 X 10(-7) M Ca2+ in the presence of phosphatidylserine (50 micrograms/ml). The effect of phosphatidylserine was biphasic. In the presence of Ca 10(-6) M, 32P incorporation increased to a maximum at 70 micrograms/ml of phosphatidylserine. The increase was suppressed at 150 micrograms/ml. Tetracaine caused a dose-dependent inhibition of calcium-activated, phospholipid-dependent enhancement of the three phosphorproteins. Calcium in the absence of phospholipid enhanced the phosphorylation of a protein of 98,000 mol wt. Phosphatidylserine suppressed this enhancement. Calmodulin (10(-6) M) had no detectable effect upon phosphorylation beyond that of calcium alone, but the calmodulin inhibitor R-24571 specificlly inhibited the calcium-stimulated 98,000 mol wt phosphoprotein. Calcium-activated, phospholipid-dependent phosphoproteins are present in human placental cytosol; whether calcium-activated, calmodulin-dependent phosphoproteins also are present remains a question.     

Identification of capacitation-associated phosphoproteins in porcine sperm electroporated with ATP-gamma-(32)P.

Our objectives were to incorporate ATP-gamma-(32)P into boar sperm to radiolabel endogenous phosphoproteins and compare phosphorylation patterns from sperm incubated in capacitating (CM) and non-capacitating conditions (NCM). Sperm were electroporated (1000 V/cm, 125 microF/cm, 65 Omega/cm, 0.3 msec) with ATP-gamma-(32)P which moderately decreased sperm viability (P < 0.01), but did not affect motility (P = 0.34) or the appearance of spontaneous acrosome reactions (P = 0.49). Sperm incubated in CM for 3 hr underwent capacitation, determined by the ability to undergo ionophore-induced acrosome reactions (P 0.05) and the 57 kDa phosphoprotein increased after capacitation (P /= 0.02). ATP-gamma-(32)P can, therefore, be incorporated into porcine sperm to radiolabel endogenous phosphoproteins, and the different profiles from sperm incubated in NCM versus CM suggest that capacitation is mediated by signaling events involving protein phosphorylation.