Phosphorylation-dependent interaction of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHA) with PADI6 following oocyte maturation in mice

Proteins in the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein family (YWHA; also known as 14-3-3) are involved in the regulation of many intracellular processes. We have examined the interaction of YWHA with peptidylarginine deiminase type VI (PADI6), an abundant protein in mammalian oocytes, eggs, and early embryos. Peptidylarginine deiminases catalyze the posttranslational modification of peptidylarginine to citrulline. PADI6 is associated with oocyte cytoplasmic sheets, and PADI6-deficient mice are infertile because of disruption of development beyond the two-cell stage. We found that PADI6 undergoes a dramatic developmental change in phosphorylation during oocyte maturation. This change in phosphorylation is linked to an interaction of PADI6 with YWHA in the mature egg. Recombinant glutathione S-transferase YWHA pull-down experiments and transgenic tandem affinity purification with liquid chromatography-mass spectrometry demonstrate a binding interaction between YWHA and PADI6 in mature eggs. YWHA proteins modulate or complement intracellular events involving phosphorylation-dependent switching or protein modification. These results indicate that phosphorylation and/or YWHA binding may serve as a means of intracellular PADI6 regulation.     

Changes in intracellular distribution and activity of protein phosphatase PP1gamma2 and its regulating proteins in spermatozoa lacking AKAP4

The second messenger cAMP mediates its intracellular effects in spermatozoa through cAMP-dependent kinase (PKA, formally known as PRKACA). The intracellular organization of PKA in spermatozoa is controlled through its association with A-kinase-anchoring proteins (AKAPs). AKAP4 (A kinase [PRKA] anchor protein 4; also called fibrous sheath component 1 or AKAP 82) is sperm specific and the major fibrous sheath protein of the principal piece of the sperm flagellum. Presumably, AKAP4 recruits PKA to the fibrous sheath and facilitates local phosphorylation to regulate flagellar function. It is also proposed to act as a scaffolding protein for signaling proteins and proteins involved in metabolism. Akap4 gene knockout mice are infertile due to the lack of sperm motility. The fibrous sheath is disrupted in spermatozoa from mutant mice. In this article, we used Akap4 gene knockout mice to study the effect of fibrous sheath disruption on the presence, subcellular distribution, and/or activity changes of PKA catalytic and regulatory subunits, sperm flagellum proteins PP1gamma2 (protein phosphatase 1, catalytic subunit, gamma isoform, formally known as PPP1CC), GSK-3 (glycogen synthase kinase-3), SP17 (sperm autoantigenic protein 17, formally known as SPA17), and other signaling proteins. There were no changes in the presence and subcellular distribution for PP1gamma2, GSK-3, hsp90 (heat shock protein 1, alpha, formally known as HSPCA), sds22 (protein phosphatase 1, regulatory [inhibitor] subunit 7, formally known as PPP1R7), 14-3-3 protein (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein), and PKB (thymoma viral proto-oncogene, also known as AKT) in mutant mice. However, the subcellular distributions for PKA catalytic subunit and regulatory subunits, PI 3-kinase (phosphatidylinositol 3-kinase), and SP17 were disrupted in mutant mice. Furthermore, there was a significant change in the activity and phosphorylation of PP1gamma2 in mutant compared with wild-type spermatozoa. These studies have identified potentially significant new roles for the fibrous sheath in regulating the activity and function of key signaling enzymes.     

Identification of a tyrosine-phosphorylated CCCTC-binding nuclear factor in capacitated mouse spermatozoa

The molecular basis of mammalian sperm capacitation, either in vivo in the female reproductive tract, or in vitro, is poorly understood. It is well known that sperm capacitation is associated with an increase in tyrosine phosphorylation of a subset of proteins. We resolved the phosphoproteins in the cell lysate of mouse sperm after capacitation by 2-DE. One tyrosine-phosphorylated 130-kDa spot was trypsin-digested, and six oligopeptide sequences were established from the MS data. These were confirmed in a CCCTC-binding nuclear factor (CTCF), a widely expressed and highly conserved protein. Further, both an anti-phosphotyrosine antibody and an anti-CTCF antibody showed immunoreactivity to a 130-kDa component in the immunoprecipitates obtained after incubation of the cell lysate from the capacitated sperm using another anti-CTCF antibody. The data support the presence of a tyrosine-phosphorylated CTCF in the capacitated sperm. Immunolocalization of the CTCF revealed fluorescent staining in the acrosome region in both capacitated and incapacitated sperm. The electrophoretic mobility shift assay, using a CTCF target sequence 5'-GGCGGCGCCGCTAGGGGTCTCTCT-3' found in the promoter of the amyloid beta-protein precursor, manifested that, relative to CTCF in the incapacitated sperm, the tyrosine-phosphorylated protein in the capacitated sperm had stronger affinity to the CTCF target sequence.     

Time-Dependent Increase in Protein Phosphorylation Following One-Trial Enhancement in Hermissenda

Abstract: One-trial conditioning of the nudibranch mollusk Hermissenda produces short- and long-term changes in excitability (enhancement) of identified sensory neurons. To investigate the biochemical mechanisms underlying this example of plasticity, we have examined changes in protein phosphorylation at different times following the in vitro conditioning trial. Changes in the incorporation of ³²PO₄ into proteins were determined using two-dimensional polyacrylamide gel electrophoresis, autoradiography, and densitometry. Conditioning resulted in increases in levels of several phosphoproteins, five of which, ranging in apparent molecular mass from 22 to 55 kDa, were chosen for analysis. The increased phosphorylation of the 46- and 55-kDa phosphoproteins detected 2 h postconditioning was significantly greater than the level of phosphorylation detected in an unpaired control group, indicating that long-term enhancement is pairing specific. Statistically significant increases in phosphorylation as compared with the control group that received only light were detected immediately after conditioning (5 min) for the 55-, 46-, and 22-kDa phosphoproteins, at 1 h for the 55- and 46-kDa phosphoproteins, and at 2 h for the 55-, 46-, and 22-kDa phosphoproteins. The 46- and 55-kDa phosphoproteins are putative structural proteins, and the 22-kDa phosphoprotein is proposed to be a protein kinase C substrate previously identified in Hermissenda following multitrial classical conditioning. Time-dependent increases in protein phosphorylation may contribute to the induction and maintenance of different memory stages expressed in sensory neurons after one-trial conditioning.     

Effects of staurosporine, okadaic acid and sodium fluoride on protein phosphorylation in graviresponding oat shoot pulvini.

This study focuses on the characterization of protein phosphorylation in the gravitropic response in oat shoot pulvini through the use of inhibitors of this process, namely staurosporine, okadaic acid and sodium fluoride. These three inhibitors reduce gravitropic curvature and cause changes in the phosphorylation of 38- and 50-kDa soluble proteins which show different levels of phosphorylation between lower and upper halves of gravistimulated pulvini. A kinetic analysis of phosphorylation shows that the 38- and 50-kDa soluble proteins exhibit different levels of phosphorylation between lower and upper halves of graviresponsive pulvini at 5 min after initiation of gravistimulation of stems. In addition, the phosphorylation of 63- and 70-kDa proteins from a total membrane preparation increases in lower halves of the pulvini following gravistimulation. These phosphoproteins are not found in the plasma membrane fraction. Taken together, at least four kinds of phosphoproteins are gravi-related. Of these, the 38- and 50-kDa soluble phosphoproteins may be involved in the regulation of early stages of the gravitropic response.     

Src family kinase-dependent phosphorylation of a 29-kDa caveolin-associated protein

PDGF receptors and Src family kinases are concentrated in caveolae, where signal transduction cascades involving these molecules are thought to be organized. The Src family tyrosine kinases are cotransducers of signals emanating from the activated PDGF receptor. However, the Src family kinase substrates that are involved in PDGF-induced signaling remain to be fully elucidated. We have identified a 29-kDa protein in caveolae that was phosphorylated in response to PDGF stimulation. This protein, pp29, was tightly bound to the caveolar coat protein caveolin-1. pp29 was among the most prominent phosphoproteins observed in cells overexpressing Fyn, suggesting that it may be a Fyn substrate. Consistent with this, pp29 was among a specific subset of proteins whose PDGF-stimulated phosphorylation was blocked by expression of kinase inactive Fyn. These data indicate that pp29 lies downstream of Fyn activation in a PDGF-stimulated signaling pathway, and that pp29 is an abundant site for nucleation of signal transduction cascades.     

Identification of capacitation associated tyrosine phosphoproteins in buffalo (Bubalus bubalis) and cattle spermatozoa

At ejaculation mammalian sperm lack fertilizing ability as they are released in a functionally immature form. The capacity to fertilize eggs is only acquired after they have been educated in the female reproductive tract and this phenomenon is termed as capacitation. Sperm capacitation includes a cascade of biochemical modifications, including cholesterol efflux, Ca(2+) influx and cAMP/PKA-dependent/independent protein tyrosine phosphorylation which is specifically considered as the biochemical marker for capacitation. The identification of tyrosine phosphoproteins shall be useful in delineating their physiological role in different events associated with sperm capacitation. The present study was conducted to identify the tyrosine phosphoproteins in the capacitated buffalo and cattle spermatozoa using 2D immunoblotting and mass spectrometry. Among several proteins identified in the buffalo capacitated sperm, serine/threonine-protein phosphatase PP1-gamma catalytic subunit, MGC157332 protein, alpha-enolase, 3-oxoacid CoA transferase 2 and actin-like protein 7A were identified as new tyrosine phosphorylation substrates in mammalian spermatozoa. Cattle sperm also contain proteins such as serine/threonine-protein phosphatase PP1-alpha catalytic subunit and membrane metallo-endopeptidase-like 1 which have not been reported as tyrosine phosphorylated in any other species. Though the presence of serine/threonine-protein phosphatase PP1-alpha catalytic subunit was demonstrated for the first time in mammalian sperm, further studies are required for its existence and possible role in different sperm functions.     

H2O2 potentiates phosphorylation of novel putative substrates for the insulin receptor kinase in intact Fao cells

Western blotting with anti-phosphotyrosine antibodies was employed in order to study insulin-dependent protein tyrosine phosphorylation in intact Fao cells. In insulin-treated cells, a prominent 180-kDa protein underwent tyrosine phosphorylation, which peaked at 45 s and then rapidly declined. Pretreatment of the cells with 1 mM Bt2cAMP or 0.16 microM 12-O-tetradecanoylphorbol-13-acetate inhibited the insulin-dependent phosphorylation of pp 180, while 1 mM vanadate or 3 mM H2O2 markedly potentiated it. These results indicate that phosphorylation of pp 180 is respectively regulated by agents that are known to synergize with or antagonize the action of the insulin receptor kinase. pp 180 is therefore likely to mediate physiological functions of this receptor kinase. Incubation of Fao cells with 3 mM H2O2 for 30 min prior to their treatment with insulin for 45 s allowed the detection of additional, previously undescribed, proteins pp 150, 114, 100, 85, 68, and 56 kDa that underwent insulin-dependent tyrosine phosphorylation. The potentiating effects of H2O2 were time- and dose-dependent and could be reversed by 2 mM dithiothreitol. Proteins phosphorylated in response to H2O2 plus insulin maintained their fully phosphorylated state for at least 20 min. We suggest that these phosphoproteins are potential physiological substrates for the insulin receptor kinase.     

The 90-kilodalton peptide of the heme-regulated eIF-2 alpha kinase has sequence similarity with the 90-kilodalton heat shock protein

Highly purified preparations of the heme-controlled eIF-2 alpha (eukaryotic peptide initiation factor 2 alpha subunit) kinase of rabbit reticulocytes contain an abundant 90-kilodalton (kDa) peptide that is immunologically cross-reactive with spectrin and that modulates the activity of the enzyme [Kudlicki, W., Fullilove, S., Read, R., Kramer, G., & Hardesty, B. (1987) J. Biol. Chem. 262, 9695-9701]. The amino-terminal sequence of the 90-kDa protein has a high degree of similarity with the known amino-terminal sequences of the Drosophila 83-kDa heat shock protein (20 out of 22 residues) and with other related heat shock proteins. The amino acid sequence of a tryptic phosphopeptide isolated by high-performance liquid chromatography from the eIF-2 alpha kinase associated 90-kDa protein after phosphorylation by casein kinase II is shown to be identical with a 14 amino acid segment of the known sequence of the Drosophila 83-kDa heat shock protein. Results of hydrodynamic studies indicate a highly elongated structure for the reticulocyte protein, characteristic of a structural protein. Additional structural similarities between the eukaryotic heat shock proteins, the reticulocyte eIF-2 alpha kinase associated 90-kDa peptide, and spectrin are discussed.     

Molecular identities of human sperm proteins that bind human zona pellucida: Nature of sperm–zona interaction,tyrosine kinase activity,and involvement of FA-1

The present study was conducted to investigate the molecular identities, nature of interaction, and tyrosine phosphorylation activity of the spermzona pellucida binding proteins in humans. Sperm proteins belcnging to four major molecular regions, namely 95, 63, 51, and 14–18 kDa, reacted with zona pellucida proteins in the Western blot and immunoprecipitation procedures. In these procedures, zona pellucida protein that reacted strongest with the sperm proteins belonged to the molecular region of 55 kDa (ZP3), besides weakly reacting proteins in the 110-kDa (ZP1/ZP2) and 14–18-kDa molecular regions. The major forces involved in the sperm-zona protein interactions were of hydrophobic and ionic in nature. Three (95, 51, and 14–18 kDa) of the four molecular regions of sperm proteins that bound to the zona pellucida proteins also seem to involve o-phospho-L-tyrosine residues in their interaction, and these proteins demonstrated the presence of phosphotyrosine residues, and the 51-kDa protein also showed autophosphorylating activity in the in vitro kinase assay. The sperm binding zona protein of 55 kDa also demonstrated autophosphorylating activity. Using specific monoclonal antibody to the well characterized sperm-specific glycoprotein, designated FA-1, and the competitive inhibition in the immunoprecipitation procedure, it was found that the 51 kDa protein is indeed FA-1 antigen. Besides elucidating the molecular nature of the spermzona interaction, these antigens will find application in the development of a multivalent contraceptive vaccine, and may also help in specific diagnosis and treatment of infertility mediated through defective gamete (sperm or oocyte) function. © 1994 Wiley-Liss, Inc.     

Stimulation of factor-dependent myeloid cell lines with interleukin 3 induces tyrosine phosphorylation of several cellular substrates

Interleukin 3 (IL-3) is required for the proliferation of growth factor-dependent myeloid cell lines. To determine the possible signal transduction mechanisms involved in IL-3 growth regulation, we have examined the effects of IL-3 on tyrosine phosphorylation. Using a monoclonal antibody against phosphotyrosine, IL-3 was found to specifically and rapidly induce tyrosine phosphorylation of cytoplasmic proteins of 70, 56, and 38 kDa and a membrane-associated glycoprotein of 140 kDa. Minor and/or variable detected phosphoproteins of 120, 85, 51, and 28 kDa were also seen. Oncogenes encoding tyrosine protein kinases abrogate the requirement of factor-dependent myeloid cells for IL-3. We therefore compared the phosphoprotein profiles of a transformed, IL-3-independent cell line with the IL-3-induced profile. In cells transformed with trk, the 56-, 51-, and 38-kDa cytoplasmic phosphoproteins were constitutively phosphorylated, whereas the 140-kDa phosphoprotein was only phosphorylated in the presence of IL-3. Taken together, these results support a role for tyrosine phosphorylation in the IL-3 signal transduction pathway and suggest that growth factor abrogation by oncogenes encoding tyrosine protein kinases may be due to the phosphorylation of substrates which are normally phosphorylated in response to IL-3.     

The role of Ca2+ and cyclic AMP in the phosphorylation of rat-liver soluble proteins by endogenous protein kinases

Rat liver soluble proteins were phosphorylated by endogenous protein kinase with [gamma-32P]ATP. Proteins were separated in dodecyl sulphate slab gels and detected with the aid of autoradiography. The relative role of cAMP-dependent, cAMP-independent and Ca2+-activated protein kinases in the phosphorylation of soluble proteins was investigated. Heat-stable inhibitor of cAMP-dependent protein kinase inhibits nearly completed the phosphorylation of seven proteins, including L-type pyruvate kinase. The phosphorylation of eight proteins is not influenced by protein kinase inhibitor. The phosphorylation of six proteins, including phosphorylase, is partially inhibited by protein kinase inhibitor. These results indicate that phosphoproteins of rat liver can be subdivided into three groups: phosphoproteins that are phosphorylated by (a) cAMP-dependent protein kinase or (b) cAMP-independent protein kinase; (c) phosphoproteins in which both cAMP-dependent and cAMP-independent protein kinase play a role in the phosphorylation. The relative phosphorylation rate of substrates for cAMP-dependent protein kinase is about 15-fold the phosphorylation rate of substrates for cAMP-independent protein kinase. The Km for ATP of cAMP-dependent protein kinase and phosphorylase kinase is 8 microM and 38 microM, respectively. Ca2+ in the micromolare range stimulates the phosphorylation of (a) phosphorylase, (b) a protein with molecular weight of 130 000 and (c) a protein with molecular weight of 15 000. The phosphate incorporation into a protein with molecular weight of 115 000 is inhibited by Ca2+. Phosphorylation of phosphorylase and the 15 000-Mr protein in the presence of 100 microM Ca2+ could be completely inhibited by trifluoperazine. It can be concluded that calmodulin is involved in the phosphorylation of at least two soluble proteins. No evidence for Ca2+-stimulated phosphorylation of subunits of glycolytic or gluconeogenic enzymes, including pyruvate kinase, was found. This indicates that it is unlikely that direct phosphorylation by Ca2+-dependent protein kinases is involved in the stimulation of gluconeogenesis by hormones that act through a cAMP-independent, Ca2+-dependent mechanism.     

Steel factor-induced tyrosine phosphorylation in murine mast cells. Common elements with IL-3-induced signal transduction pathways.

The c-kit/W gene encodes a transmembrane protein tyrosine kinase, which is the receptor for Steel factor (SLF). SLF shares many general characteristics of hemopoietic growth factors, stimulating the survival, proliferation, and differentiation of stem and progenitor cells. We have investigated the tyrosine phosphorylation events that ensue after SLF binding to the c-kit protein using primary cultures of murine mast cells as a model system and have compared the effects of SLF and IL-3. Proteins that became phosphorylated on tyrosine after treatment of cells with SLF included c-kit itself, and major protein substrates designated p130, p122, p118, p115, p112, p100, p77, p55, p44, and p42. The majority of these proteins were cytosolic and maximally phosphorylated within 2 min of growth factor treatment. Combinations of immunoprecipitation and immunoblotting with antibodies specific for proteins known to be associated with signaling pathways demonstrated that none of the major tyrosine-phosphorylated species correlated with phospholipase C-gamma 1, GTPase activating protein, or phosphatidylinositol 3' kinase. However, stimulation with SLF led to a modest increase in tyrosine phosphorylation of the 85-kDa subunit of the phosphatidylinositol 3' kinase and increased association with a 150-kDa phosphotyrosyl protein, likely to be c-kit. Two species that did correlate with known elements were the 44- and 42-kDa polypeptides, shown to be members of the mitogen-activated protein kinase family. A subset of these proteins (p130, p115/112, p100, p55, p44, p42) were also tyrosine-phosphorylated when cells were stimulated by IL-3. MonoQ ion-exchange chromatography and two dimensional gel analyses were used to demonstrate that at least the p55, p44, and p42 substrates were identical, as well as some more minor species of molecular weights 50, 38, and 36 kDa, thus indicating common pathways of signaling in hemopoietic cells. Whereas in the case of SLF the dose-response characteristics of the proliferative response and the induction of tyrosine phosphorylation were similar, in the case of IL-3, much lower concentrations were required for maximal proliferation than maximal tyrosine phosphorylation. These studies form the basis for further molecular characterization of common components of signal transduction pathways in hemopoietic cells.     

Progesterone and cAMP-dependent protein kinase regulate in vivo the level of phosphorylation of two proteins (Mr 20,000 and Mr 32,000) in Xenopus oocytes

The [32P]phosphoproteins and [35S]thiophosphoproteins were analyzed by electrophoresis and autoradiography after microinjection of [gamma-32P]ATP or of [35S]ATP-gamma-S into living Xenopus oocytes. The level of 32P incorporation into a 20-kDA protein was decreased following progesterone treatment (between 1 and 2 hr). This 20-kDa protein was partially thiophosphorylated in vivo by [35S]ATP-gamma-S. Furthermore it was found that this phosphoprotein was partially purified by TCA (1%) extraction and heat treatment. Microinjection of the C-subunit of cAMP-dependent protein kinase (0.6 to 1.2 pmole) inhibited maturation and provoked an increase in the level of phosphorylation of the 20-kDa protein and of a 32-kDa protein, indicating that both proteins were in vivo substrates (directly or indirectly) for cAMP-dependent protein kinase. When inhibitor-1 of protein phosphatase-1 was microinjected (5 to 10 pmole per oocyte) meiotic maturation was inhibited and the level of phosphorylation of the 32-kDa protein was increased; the same result was obtained following ATP-gamma-S (1 mM) microinjection. Altogether these results suggest that a 20-kDa phosphoprotein, whose level of phosphorylation is decreased by progesterone, could be involved in the regulation of maturation by lowering the level phosphorylation of a 32-kDa phosphoprotein. An attractive hypothesis would be that the 20-kDa phosphoprotein is an inhibitor of protein phosphatase-1.     

Mitogen-induced tyrosine-phosphorylated 41- and 43-kDa proteins are family members of extracellular signal-regulated kinases/microtubule-associated protein 2 kinases.

Two antipeptide antibodies, one against the peptide corresponding to residues 307-327 (alpha Y91) and one against the peptide corresponding to the C-terminal portion (alpha C92) of the deduced amino acid sequence of the extracellular signal-regulated kinase 1 (ERK1), precipitated two 41-kDa and/or two 43-kDa phospho-proteins from mitogen-stimulated Swiss 3T3 cells. Electrophoretic mobilities on two-dimensional gels of the immunoprecipitated 41- and 43-kDa phosphoproteins were similar to those of the 41- and 43-kDa cytosol proteins, whose increased tyrosine phosphorylation we and others had originally identified in various mitogen-stimulated cells (Cooper, J. A., Sefton, B. M., and Hunter, T. (1984) Mol. Cell. Biol. 4, 30-37; Kohno, M. (1985) J. Biol. Chem. 260, 1771-1779); phosphopeptide map analysis revealed that they were respectively identical molecules. All those phosphoproteins contained phosphotyrosine, and the more acidic forms contained additional phosphothreonine. Immunoprecipitated 41- and 43-kDa phosphoproteins had serine/threonine kinase activity toward myelin basic protein (MBP) and microtuble-associated protein 2 (MAP2). With the combination of two-dimensional gel electrophoresis and the kinase assay in MBP-containing polyacrylamide gels of the alpha Y91 immunoprecipitates, with or without phosphatase 2A treatment, we showed that only their acidic forms were active. These results clearly indicate that 41- and 43-kDa proteins, the increased tyrosine phosphorylation of which is rapidly and commonly induced by mitogen stimulation of fibroblasts, are family members of ERKs/MAP2 kinases and that phosphorylation both on tyrosine and threonine residues is necessary for their activation.     

Interleukin 1 and tumour necrosis factor increase phosphorylation of the small heat shock protein. Effects in fibroblasts, Hep G2 and U937 cells

Interleukin 1 alpha and tumour necrosis factor-alpha stimulated phosphorylation of three 27 kDa phosphoproteins in MRC-5 fibroblasts which was sustained for up to 2 h after adding the cytokines. All three phosphoproteins were immunoprecipitated by a specific antiserum to the small mammalian heat shock protein, hsp 27. The three phosphoproteins from stimulated or control cells contained phosphoserine but not phosphothreonine or phosphotyrosine. Similar increases in phosphorylation of immunoprecipitable 27 kDa proteins were seen in U937 cells stimulated by TNF alpha and Hep G2 cells stimulated by IL1 alpha.     

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.     

Role of tyrosine phosphorylation of flagellar proteins in hamster sperm hyperactivation.

Despite extensive study of sperm motility, little is known of the mechanism of mammalian sperm hyperactivation. Here we describe a novel method for preparation of rodent sperm flagella and use it to show a correlation between tyrosine phosphorylation of flagellar proteins and hyperactivation of hamster sperm. When hyperactivation was produced by a 3.5-h incubation in a medium supporting capacitation, four major tyrosine-phosphorylated peptides of 90-, 80-, 62-, and 48-kDa mass were detected in flagellar extracts. Incubation with calyculin A, an inhibitor of protein phosphatases 1 and 2A, produced hyperactivation within 40 min but only a single 80-kDa phosphotyrosine-containing flagellar component. Conversely, incubation with inhibitors of either protein kinase A (H8) or protein tyrosine kinase (tyrphostin 47) prevented both hyperactivation and the production of tyrosine-phosphorylated flagellar peptides. These results indicate a strong correlation of hyperactivation with the tyrosine phosphorylation of sperm flagellar peptides, and they strongly implicate an 80-kDa component as a major mediator of the mechanism that produces hyperactivated motility of hamster sperm.     

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.