Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We isolated and characterized several monoclonal antibodies against a protein complex containing the flagellar movement-initiating phosphoprotein (MIPP) that appears to play a crucial role in the initiation of flagellar movement in quiescent spermatozoa of Salmonid fish. The effects of the antibodies on the phosphorylation of MIPP, as well as on the initiation of movement, in model sperm cells were studied. Three monoclonal antibodies, namely, FMI7, FMI18, and FMI27, were found specifically to inhibit both the initiation of flagellar movement and the phosphorylation of MIPP. These antibodies did not recognize denatured MIPP; they only recognized the native antigen. FMI7 exclusively recognized the denatured form of a 38-kDa protein, which may possibly be a protein kinase responsible for the phosphorylation of MIPP. Immunofluorescence analysis in situ of model sperm cells with the antibodies showed that the antigen was localized predominantly in the basal structure of the spermatozoon. Thus, the results clearly demonstrate the involvement of MIPP in the initiation of flagellar movement and the control of flagellar motility.
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PMID:Monoclonal antibodies against the protein complex that contains the flagellar movement-initiating phosphoprotein of Oncorhynchus keta. 796 3

The majority of cellular responses to changing environmental conditions is regulated by protein kinases. Spermatozoa have many special properties, including motility with demonstrated chemotaxis, the ability to undergo capacitation, and the acrosome reaction, which are in part controlled by extracellular signals and in which sperm kinases are considered to be involved. We have previously reported that there is a protein kinase activity, which phosphorylates the synthetic substrate poly-(Glu, Tyr) with a Km value of 2.3 microM, and is inhibited by the tyrosine kinase inhibitor tyrphostin, in the protein extract from boar spermatozoa (Berruti and Porzio, 1992: Biochim Biophys Acta 1118:149-154). Now we have demonstrated that the enzyme is cytosolic, is active as a monomer of M(r) 42,000, is stimulated by Mg2+ > Mn2+ but not by Ca2+, is renaturable, and can phosphorylate native protein substrates such as microtubule-associated protein 2 (MAP2) and histone H2B both on the tyrosine and serine residues. N-terminal sequence analysis suggests that it is a novel protein. These new findings imply that the boar sperm 42 kD kinase may be a novel member of the emerging class of dual-specificity protein kinases, and they raise the intriguing question of its function in the protein kinase network mediating signal transduction in mammalian spermatozoa.
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PMID:Biochemical characterization of the boar sperm 42 kilodalton protein tyrosine kinase: its potential for tyrosine as well as serine phosphorylation towards microtubule-associated protein 2 and histone H 2B. 798 Sep 47

P1 protamines isolated from ejaculated human, stallion, bull, boar and ram spermatozoa and P2 protamines from human and stallion spermatozoa were subjected, after alkaline phosphatase treatment, to in vitro phosphorylation reactions using cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). All P1 protamines were phosphorylated by PKA, whereas P2 protamines were phosphorylated only by PKC. In addition, human, stallion and boar, but not bull and ram, P1 protamines were phosphorylated by PKC. After phosphoamino acid analysis, the protamines showing positive signals for phosphoserine (P-Ser) were subjected to P-Ser conversion reaction and protein sequencing. Only stallion (St1) and human (HP1) P1 protamines contained P-Ser after PKA phosphorylation, located in the middle region of the molecule, i.e., at Ser29 in St1 and Ser28 in HP1. All other phosphorylated P1 protamines contained only P-Thr, which could not be further localized in the sequence with the present methods. After PKC phosphorylation, the internally located Ser residues in human (ser21) and stallion (Ser29) P1 protamines were phosphorylated and, in boar P1 protamine, only Thr43 was slightly phosphorylated. The N-terminally located Ser residues in P1 protamines, which are known to be phosphorylated in vivo, were not phosphorylated by either kinase, indicating that there must still be other types of protamine kinases in sperm cells responsible for their phosphorylation. Within P2 protamines, HP2 was equally well phosphorylated at all Ser residues in addition to some Thr phosphorylation, whereas, in St2, Ser32 was the main target for PKC phosphorylation in vitro. Collectively, PKC is a good candidate for in vivo phosphorylation of P2 protamines and PKA for phosphorylation of some hydroxyamino acid residues in P1 protamines.
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PMID:P2 protamines are phosphorylated in vitro by protein kinase C, whereas P1 protamines prefer cAMP-dependent protein kinase. A comparative study of five mammalian species. 803 90

A variety of fatty acids including the cis-polyunsaturated very-long-chain fatty acids (VLCFA) (> 22 carbon atoms) common in retina, spermatozoa, and brain were examined for their ability to activate protein kinase C (PKC) purified from rat brain. Arachidonic [20:4(n-6)], eicosapentaenoic [20:5(n-3)], and docosahexaenoic [22:6(n-3)] acids as well as the VLCFA dotriacontatetraenoic [32:4(n-6)] and tetratriacontahexaenoic [34:6(n-3)] were equally capable of activating PKC in vitro with maximal activity being between 25 and 50 microM. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate further enhanced the in vitro activation of PKC when added to the protein kinase assay system with the fatty acids. The fully saturated arachidic acid (20:0) was inactive in both assay systems. The potential significance of the in vitro activation of PKC by the VLCFA is discussed.
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PMID:In vitro activation of rat brain protein kinase C by polyenoic very-long-chain fatty acids. 813 82

Cyclic AMP-dependent changes in phosphorylation of epididymal mouse sperm suspensions were examined in media designed to manipulate capacitation and the expression of parameters associated with full fertilizing ability, i.e. hyperactivated motility and the acrosome reaction. After initial assessment of cAMP-dependent protein kinase activity in frozen-thawed and lyophilized sperm suspensions using exogenous substrate, phosphorylation of endogenous sperm phosphoproteins was examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by autoradiography or immunoblotting. Numerous phosphoproteins were detected in both incapacitated and capacitated suspensions, the majority of which were probably concerned with motility; full expression of fertilizing ability appeared to involve an increase in the amount of endogenous phosphorylation as deduced from the decreased amount of 32P incorporation in these suspensions. The addition of the cAMP-dependent protein kinase inhibitors, H8 and PKI (6-22) amide, demonstrated that most of the phosphoproteins detected were phosphorylated in a cAMP-dependent manner. Of particular interest was a phosphoprotein with an M(r) of about 95,000 which was consistently observed in capacitated suspensions. Evidence suggests that this may be phosphorylated on tyrosine residues, since the inclusion of orthovanadate, a phosphoryltyrosine phosphatase inhibitor, altered phosphorylation of this protein. Furthermore, immunodetection using the antiphosphotyrosine antibody, PY-20, identified five proteins with approximate M(r) 116,000, 105,000, 95,000, 86,000, and 76,000, and possibly a sixth at 54,000. The 95,000 protein was consistently diminished in ionophore-treated spermatozoa, indicating that the protein was located in the acrosomal cap region. These results suggest that the protein may be the same phosphotyrosine-containing protein as that described by Leyton and Saling (1989) which has been proposed to play a role in acrosomal exocytosis.
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PMID:Cyclic AMP-dependent phosphorylation of epididymal mouse sperm proteins during capacitation in vitro: identification of an M(r) 95,000 phosphotyrosine-containing protein. 838 23

The osmo-sensitivity of the human sperm acrosome reaction was investigated by determining the effect of hyper- and hypo-osmolal conditions on the ionophore A23187- and dbcAMP-induced reaction of both capacitated and non-capacitated spermatozoa. Hyper-osmolal conditions inhibited the agonist-induced reactions of both types of spermatozoa. Hypo-osmolal conditions caused a spontaneous loss of acrosomes from capacitated but not from non-capacitated spermatozoa. The loss of acrosomes under hypo-osmolal conditions was further enhanced by dbcAMP but not by ionophore A23187. Although significant decreases in sperm viability were occasionally observed at the high and low osmolalities, these decreases were not consistent and could not account for the observed loss of acrosomes. It is concluded that the human sperm acrosome reaction is osmo-sensitive. The acrosome reaction stimulated by ionophore A23187 (raises intracellular Ca2+) and dbcAMP (activates protein kinase A which causes protein phosphorylation) appears to involve water entry downstream from the action of these agonists. Preincubation in albumin (capacitation) causes human spermatozoa to lose their acrosomes under hypo-osmolal conditions. Finally, capacitation is not an essential prerequisite to the acrosome reaction as long as agonists are used that by-pass certain membrane-related events.
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PMID:Osmo-sensitivity of the human sperm acrosome reaction. 840 20

In order to minimize the percentage of false-negative results in the zona-free sperm penetration assay (SPA), a wide range of substances and/or physical agents capable of inducing the acrosome reaction (AR) have been incorporated in the incubation medium. These agents can also be used for treatment of severe male infertility using the technique of sperm microinjection under the zona pellucida (SMUZ). In the present review, the percentages of acrosome-reacted spermatozoa induced by several physiological, biochemical or physical agents published in the literature are compared in order to find the most efficient method(s) of inducing the AR in human sperm as a previous requirement of optimizing the technique of SMUZ. A working estimate of the level of efficiency of a given AR inducer is calculated by adding up its range score in each of three different arrangements from the highest to the lowest value of percentages of AR and differences in percentages of AR and penetration indexes between treated and control groups in SPA. The agents able to induce the AR by nonphysiological (electropermeabilization, lysophosphatidyl choline, and freezing-thawing) have better positions in this hierarchical system than those ones which require the active participation of sperm membrane receptors or second messenger systems (progesterone, zona pellucida, and stimulators of protein kinase A). Electropermeabilization appears to be the most efficient AR inducer. However, more possibilities need to be explored to enhance the relatively low percentages of acrosome-reacted spermatozoa shown by infertile men.
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PMID:Zona-free sperm penetration assay and inducers of the acrosome reaction: a model for sperm microinjection under the zona pellucida. 850 86

A specific peptide inhibitor of the cyclic AMP (cAMP)-dependent protein kinase (PKI-peptide) is a very effective inhibitor of the cAMP-dependent activation of motility of Ciona spermatozoa, when PKI-peptide is present at the beginning of incubation of demembranated spermatozoa with cAMP and ATP. Under conditions where approximately 120 sec is required for full activation of motility, the window of sensitivity to the PKI-peptide lasts for only 25-30 sec. Examination of sperm pellet proteins labeled with 32P ATP during activation reveals a major 25 kDa phosphoprotein and 2 minor phosphoproteins whose phosphorylation is highly sensitive to to inhibition by the PKI-peptide and essentially complete during this early phase. These sperm proteins appear to be immediate substrates for cAMP-dependent protein kinase, and phosphorylation of one or more of these appears to be requires, but not sufficient, for activation of motility. The phosphorylation of other proteins is reduced or eliminated when PKI-peptide is present at the beginning of incubation, but is unaffected by later addition of PKI-peptide. Some of these substrates appear to be likely candidates for axonemal proteins that must be phosphorylated during the later stages of incubation in order to complete the activation process. This selection is based upon a high degree of inhibition by inclusion of PKI-peptide or other inhibitors at the start of the incubation process, on near-completion of their phosphorylation by the end of the 2 min incubation period required for the activation of motility, and evidence that these proteins are phosphorylated during in vivo activation of motility. Although these observations suggest the presence of a second kinase activity that is upregulated by the initial activation of the cAMP-dependent protein kinase, assays using exogenous substrates have not yet been able to identify such a kinase activity.
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PMID:Multiple protein kinase activities required for activation of sperm flagellar motility. 867 35

The effects of an adenosine agonist with specificity for A2 receptors, on human sperm prepared for in vitro fertilization (IVF), were examined to verify physiological effects and possible pharmacological use. 5' -N-ethyl-carboxamidoadenosine (NECA), when added at 100 microM over 30 min in B2 medium, did not induce a spontaneous acrosome reaction after 0, 3, and 6 h capacitation in B2, nor did it modify sperm motility. However, NECA increased the number of capacitated spermatozoa able to respond (p < 0.05) to A23187 (10 microM) after 6 h preincubation in B2 medium. This effect was associated with an increase in cAMP production, which was measured by RIA after 10 and 20 min incubation with NECA in uncapacitated sperm, and with changes in the kinetics of protein tyrosine phosphorylation as revealed by Western blot. Phosphorylation of a 95-kDa protein was enhanced by NECA in uncapacitated sperm and inhibited in capacitating sperm (incubated 1 h in B2), whereas phosphorylation of a 50-kDa protein was systematically enhanced whatever the preincubation time. NECA can stimulate uncapacitated human sperm via cAMP production and protein phosphorylation/dephosphorylation without inducing an acrosome reaction or influencing motility. Cyclic AMP-dependent protein kinase A seems to positively control protein phosphorylation involved in human capacitation. Adenosine present in the tubal fluid or produced by the spermatozoon itself may influence capacitation in vivo through sperm A2 receptors. In cases of male infertility, use of NECA in sperm handling for IVF should be evaluated as a means to improve capacitation without increasing the possibility of a premature spontaneous acrosome reaction.
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PMID:Stimulation of human sperm during capacitation in vitro by an adenosine agonist with specificity for A2 receptors. 872 71

Acrosomal exocytosis triggered with A23187/Ca2+ was enhanced by rab3AL, a synthetic peptide corresponding to the effector domain of the small GTP-binding protein rab3. Exocytosis was further enhanced when spermatozoa were also exposed to dibutyryl-cAMP, but was prevented when H-89, a protein kinase A (PKA) inhibitor, was included. The action of rab3AL was not on, or upstream of, phospholipase A2 (PLA2). Inhibition of exocytosis by the PLA2 inhibitor aristolochic acid was overcome by rab3AL when it was included together with lysophosphatidylcholine; this effect was prevented by H-89. These results suggest a functional coupling between rab3 protein, metabolites generated by PLA2, and cAMP-activated PKA, in the final steps leading to membrane fusion during acrosomal exocytosis.
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PMID:rab 3-peptide stimulates exocytosis of the ram sperm acrosome via interaction with cyclic AMP and phospholipase A2 metabolites. 876 86


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