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)

Signal transduction pathways regulate various aspects of mammalian sperm function. When human sperm were incubated in a medium supporting capacitation, proteins became tyrosine-phosphorylated in a time-dependent manner. This phosphorylation was inhibited by genistein, a protein tyrosine kinase inhibitor. Phosphorylation was also reduced when sperm were incubated either in the presence of increasing concentrations of extracellular Ca2+ or in a medium containing the Ca2+ ionophore A23187. This Ca2+-induced dephosphorylation was calmodulin-dependent, suggesting that calcineurin was involved. In this regard, the calcineurin inhibitor deltamethrin inhibited the Ca2+ ionophore-induced dephosphorylation. A limited number of Mr 80,000-105,000 polypeptides were the most prominent phosphotyrosine-containing proteins present in human sperm. Unlike mouse sperm, which contains a tyrosine-phosphorylated isoform of hexokinase, a phosphotyrosine-containing hexokinase in human sperm was not detected. Most of the tyrosine-phosphorylated proteins were Triton X-100-insoluble and were localized to the principal piece of the flagellum, the region where the cytoskeletal fibrous sheath is found. Prominent phosphotyrosine-containing proteins of Mr 82,000 and 97,000 were identified as the human homologues of mouse sperm AKAP82, the major fibrous sheath protein, and pro-AKAP82, its precursor polypeptide, respectively. These proteins are A Kinase Anchor Proteins, polypeptides that sequester protein kinase A to subcellular locations. Taken together, these results suggest that protein tyrosine phosphorylation may be part of a signal transduction cascade(s) regulating events pertaining to capacitation and/or motility in mammalian sperm and that an interrelationship between tyrosine kinase and cAMP signaling pathways exists in these cells.
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PMID:Regulation of protein tyrosine phosphorylation in human sperm by a calcium/calmodulin-dependent mechanism: identification of A kinase anchor proteins as major substrates for tyrosine phosphorylation. 894 91

The assembly of the mammalian sperm flagellum is a complex developmental event requiring the sequential activation of genes encoding the component parts and the coordinated assembly of these proteins during the differentiation of the haploid spermatid. In this study, the mechanism underlying the assembly of the fibrous sheath surrounding the axoneme was examined. The subject of the study was the major fibrous sheath protein of the mouse sperm flagellum, AKAP82, a member of the A Kinase Anchor Protein (AKAP) family of polypeptides that bind the regulatory (RII) subunit of protein kinase A (PK-A). Immunoelectron microscopy demonstrated that AKAP82 is present throughout the transverse ribs and longitudinal columns of the fibrous sheath. Since AKAP82 is initially synthesized as a precursor (pro-AKAP82) during spermiogenesis, an antiserum was raised against a peptide from the processed region of pro-AKAP82 (M(r) 97,000). In immunoblotting experiments, the antibody detected pro-AKAP82 in condensing spermatids but not in epididymal sperm. In addition, two other immunoreactive proteins of M(r) 109,000 (p109) and M(r) 26,000 (p26, representing the "pro" domain of the precursor) were present in epididymal sperm. Alkaline phosphatase treatment of epididymal sperm proteins demonstrated that p109 was a phosphorylated form of pro-AKAP82 that remained in sperm. By immunofluorescence, pro-AKAP82 was localized to the entire length of the principal piece in testicular sperm, while in epididymal sperm p109 and p26 were present only in the proximal portion of the principal piece. Pro-AKAP82 was solubilized when germ cells were extracted with Triton X-100. However, in sperm, both AKAP82 and p109 were almost totally resistant to these extraction conditions and remained in the particulate fraction even after extraction with Triton and dithiothreitol. Similar to pro-AKAP82, the RII subunit of PK-A was present in the Triton X-100-soluble fraction of developing germ cells. In sperm, much of the RII also became particulate, consistent with the hypothesis that AKAP82 anchors RII in the flagellum. These data indicate that pro-AKAP82 is synthesized in the cell body, transported down the axoneme to its site of assembly in the fibrous sheath, and then proteolytically clipped to form mature AKAP82.
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PMID:Assembly of AKAP82, a protein kinase A anchor protein, into the fibrous sheath of mouse sperm. 944 72

The molecular basis of mammalian sperm capacitation, defined as those biochemical and functional changes that render the sperm competent to fertilize the egg, is poorly understood. This extratesticular maturational process is accompanied by the activation of a unique signal transduction pathway involving the cAMP-dependent up-regulation of protein tyrosine phosphorylation presumably through the activation of protein kinase A (PK-A). We demonstrate in this report that capacitation of cauda epididymal mouse sperm in vitro was accompanied by a time-dependent increase in PK-A activity. This increase in PK-A activity did not occur in a medium that does not support capacitation. While PK-A catalytic and RI/RII regulatory subunits, as well as PK-A enzyme activity, were found in both the Triton X-100-soluble and -insoluble fractions of the sperm, the increase in PK-A activity accompanying capacitation was associated with enzyme activity found in the soluble fraction. Moreover, the regulatory and catalytic subunits of PK-A were observed by indirect immunofluorescence to be present throughout the head, midpiece, and principal piece of the sperm. Thus, PK-A appears to be functional in multiple compartments of this highly differentiated cell. A fraction of the Triton X-100-insoluble PK-A is presumably tethered by AKAP82, the major protein of the fibrous sheath of the sperm flagellum which anchors and compartmentalizes PK-A to the cytoskeleton via the RII subunit of PK-A. Using various recombinant truncated AKAP82 constructs in a gel overlay assay, the RII subunit-binding domain of this protein was mapped to a 57-amino-acid residue region at its N-terminus. Computer analysis revealed a 14-amino-acid region that resembled the RII-binding domains of other A Kinase Anchor Proteins. A synthetic peptide corresponding to this domain inhibited AKAP82-RII binding in a gel overlay assay, providing further support that AKAP82 is an anchoring protein for the subcellular localization of PK-A in the mouse sperm fibrous sheath. This work, along with previous findings that cAMP is a key intermediary second messenger in regulating protein tyrosine phosphorylation and capacitation, further supports the importance of PK-A in these processes and necessitates a further understanding of the contribution of both the soluble and insoluble forms of PK-A, as well as AKAP82, to sperm function.
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PMID:Regulation, localization, and anchoring of protein kinase A subunits during mouse sperm capacitation. 944 73

Mammalian sperm motility is regulated by a cascade of cAMP-dependent protein phosphorylation events mediated by protein kinase A. A-kinase anchor proteins (AKAPs) direct protein kinase A activity by tethering the enzyme near its physiological substrates. We have characterized a major human sperm fibrous sheath AKAP, hAKAP82, and its precursor, pro-hAKAP82, the homologues of the mouse fibrous sheath proteins mAKAP82 and pro-mAKAP82. The cDNA sequence of pro-hAKAP82 was highly homologous to the mouse sequence, and the functional domains of the pro-hAKAP82 protein, the protein kinase A binding, and the pro-hAKAP82/hAKAP82 cleavage sites were identical to those of the mouse protein. The genomic organization of mouse pro-AKAP82 was determined. Alternative splicing occurred in both the mouse and human pro-AKAP82 genes that resulted in at least two distinct transcripts and possibly two different proteins. Compared with pro-mAKAP82, considerably less pro-hAKAP82 was processed to hAKAP82 in human sperm. Although pro-mAKAP82 localizes only to the proximal portion of the principal piece of the flagellum, pro-hAKAP82 localized to the entire length of the principal piece. The pro-hAKAP82 gene mapped to human chromosome Xp11.2, indicating that defects in this gene are maternally inherited. These studies suggest several roles for hAKAP82 in sperm motility, including the regulation of signal transduction pathways.
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PMID:An X-linked gene encodes a major human sperm fibrous sheath protein, hAKAP82. Genomic organization, protein kinase A-RII binding, and distribution of the precursor in the sperm tail. 982 90

The fibrous sheath is a unique cytoskeletal structure in the sperm flagellum believed to modulate sperm motility. FSC1 is the major structural protein of the fibrous sheath. The yeast two-hybrid system was used to identify other proteins that contribute to the structure of the fibrous sheath or participate in sperm motility. When FSC1 was used as the bait to screen a mouse testis cDNA library, two clones were isolated encoding the type Ialpha regulatory subunit (RIalpha) of cAMP-dependent protein kinase. Deletion analysis using the yeast two-hybrid system and in vitro binding assays with glutathione S-transferase-FSC1 fusion proteins identified two RIalpha tethering domains on FSC1. A domain located at residues 219-232 (termed domain A) corresponds to the reported tethering domain for a type II regulatory subunit (RII) of cAMP-dependent protein kinase, indicating that this binding domain has dual specificity to RI and RII. Another RIalpha tethering site (termed domain B) at residues 335-344 shows specific binding of RIalpha and had no significant sequence homology with known RII tethering domains. However, helical wheel projection analysis indicates that domain B is likely to form an amphipathic helix, the secondary structure of RII tethering domains of protein kinase A anchoring proteins. This was supported by the finding that site-directed mutagenesis to disrupt the amphipathic helix eliminated RIalpha binding. This is apparently the first report of an RIalpha-specific protein kinase A anchoring protein tethering domain.
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PMID:Identification of tethering domains for protein kinase A type Ialpha regulatory subunits on sperm fibrous sheath protein FSC1. 985 4

Protein kinase A regulates sperm motility through the cAMP-dependent phosphorylation of proteins. One mechanism to direct the activity of the kinase is to localize it near its protein substrates through the use of anchoring proteins. A-Kinase anchoring proteins (AKAPs) act by binding the type II regulatory subunit of protein kinase A and tethering it to a cellular organelle or cytoskeletal element. We showed previously that mAKAP82, the major protein of the fibrous sheath of the mouse sperm flagellum, is an AKAP. The available evidence indicates that protein kinase A is compartmentalized to the fibrous sheath by binding mAKAP82. To characterize AKAP82 in bovine sperm, a testicular cDNA library was constructed and used to isolate a clone encoding bAKAP82, the bovine homologue. Sequence analysis showed that the primary structure of bAKAP82 was highly conserved. In particular, the amino acid sequence corresponding to the region of mAKAP82 responsible for binding the regulatory subunit of protein kinase A was identical in the bull. Bovine AKAP82 was present in both epididymal and ejaculated sperm and was localized to the entire principal piece of the flagellum, the region in which the fibrous sheath is located. Finally, bAKAP82 bound the regulatory subunit of protein kinase A. These data support the idea that bAKAP82 functions as an anchoring protein for the subcellular localization of protein kinase A in the flagellum.
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PMID:Conservation and function of a bovine sperm A-kinase anchor protein homologous to mouse AKAP82. 1041 9

Sperm motility is regulated by the cAMP-dependent protein kinase (protein kinase-A)-mediated phosphorylation of a group of largely unidentified flagellar proteins. Human AKAP82 (hAKAP82) and its precursor protein, pro-hAKAP82, are members of the A-kinase anchor protein (AKAP) family. These proteins tether protein kinase-A to the fibrous sheath of human spermatozoa and presumably localize the activity of the kinase near specific targets in the sperm flagellum. In this way, pro-hAKAP82 and hAKAP82 may be involved in regulating sperm motility. Similar to its homologues in other species, pro-hAKAP82 is proteolytically processed to hAKAP82. However, the amount of processing of pro-hAKAP82 in human spermatozoa is less than the amount of processing of the precursor in other species. We postulated that this lower extent of processing may be related to lower percentages of human sperm motility. In addition, both pro-hAKAP82 and hAKAP82 are tyrosine phosphorylated in a capacitation-dependent manner. Since capacitation is associated with hyperactivated motility, we postulated that tyrosine phosphorylation of pro-hAKAP82/hAKAP82 is associated with changes in motility. However, using a combination of immunofluorescence and immunoblotting approaches, we found no evidence for an association between either processing or tyrosine phosphorylation of pro-hAKAP82/hAKAP82 and significant differences in motility in spermatozoa from normal men.
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PMID:Relationship between sperm motility and the processing and tyrosine phosphorylation of two human sperm fibrous sheath proteins, pro-hAKAP82 and hAKAP82. 1046 Feb 19

Cyclic AMP-dependent protein kinase is tethered to protein kinase A anchoring proteins (AKAPs) through regulatory subunits (R) by RIalpha-specific, RIIalpha-specific, or RIalpha/RIIalpha dual-specific binding. Ala- and Val-scanning mutagenesis determined that hydrophobic amino acids at three homologous positions are required for binding of RIalpha to FSC1/AKAP82 domain B and RIIalpha to AKAP Ht31. A mutation at the middle position reversed the binding specificity of both AKAPs, and mutations at this same position of the dual-specific domain A of FSC1/AKAP82 converted it into either an RIalpha or RIIalpha binding domain. This suggests that hydrophobic amino acids at three conserved positions within the primary sequence and an amphipathic helix of AKAPs are required for cyclic AMP-dependent protein kinase binding, with the size of the aliphatic side chain at the middle position determining RIalpha or RIIalpha binding specificity.
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PMID:Single amino acids determine specificity of binding of protein kinase A regulatory subunits by protein kinase A anchoring proteins. 1050 57

Protein tyrosine phosphorylation has been associated with both capacitation and motility of mammalian sperm. During capacitation, human spermatozoa undergo tyrosine phosphorylation of a characteristic set of proteins, only one of which has thus far been cloned and localized. We report here the sequence of a fibrous sheath protein of 95 kDa (FSP95) that undergoes tyrosine phosphorylation during capacitation of human spermatozoa and has similarity to sperm A-kinase anchor proteins (AKAPs). FSP95 is both auto- and iso-antigenic in humans as it is recognized by sera containing antisperm antibodies from infertile men and women. The 853-residue protein has a calculated molecular weight of 94.6 kDa and an isoelectric point (pI) of 6.0, and it contains multiple potential phosphorylation sites for protein kinase C and casein kinase II as well as one potential tyrosine kinase phosphorylation site at amino acid 435. The sequence has amino acid homology to mouse sperm fibrous sheath AKAP82 (pro-mAKAP82, 34% identity) and to human sperm fibrous sheath AKAP82 (pro-hAKAP82, 32% identity). The gene encoding FSP95 has 5 exons separated by 4 introns and is located on chromosome 12 at locus p13.3. Northern analysis detected a single transcript of approximately 3.0 kilobases, and Northern dot blot analysis of 50 human tissues revealed FSP95 mRNA expression only in testis. By employing sperm immobilization, indirect immunofluorescence, and immunoelectron microscopy with antisera to purified recombinant FSP95, the protein was localized to the ribs of the fibrous sheath in the principal piece of the sperm tail. FSP95 is the second fibrous sheath protein to be cloned, sequenced and localized in human spermatozoa.
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PMID:FSP95, a testis-specific 95-kilodalton fibrous sheath antigen that undergoes tyrosine phosphorylation in capacitated human spermatozoa. 1052 64

The molecular basis of mammalian sperm capacitation is unique in that, it is associated with a protein kinase A (PKA) dependent upregulation of protein tyrosine phosphorylation. Therefore, PKA activity during capacitation would be crucial for the downstream events of protein tyrosine phosphorylation, and mechanisms may exist to ensure that PKA phosphorylates its specific substrate. This could be achieved by bringing PKA close to its substrate, a function normally carried out by an A-kinase anchoring protein (AKAP). We showed previously that cauda epididymidal spermatozoa of hamster undergo a capacitation-dependent increase in protein tyrosine phosphorylation. In the present study, evidence is provided that two major tyrosine phosphorylated proteins of molecular weight 97 and 83 kDa are the hamster homologues of mouse pro-AKAP82 and AKAP82, and have been designated as hamster pro-AKAP83 and AKAP83 respectively. Hamster AKAP83 resembled the mouse AKAP82 with respect to its molecular weight, pI (pH 5-5.5) and cDNA and amino acid sequences. Sequence analysis indicated that the primary structure of pro-AKAP83 was highly conserved and exhibited 91% identity with mouse and rat AKAP82. Further, the functional domains, namely the region involved in binding the regulatory subunit of PKA and the proteolytic cleavage site between pro-AKAP83 and AKAP83, were identical with that observed in rat and mouse pro-AKAP82 and AKAP82. Immunoblot analysis using polyclonal hamster anti-AKAP83 antibodies indicated that AKAP83 was present both in caput and cauda epididymidal spermatozoa. The antibody also identified the pro-AKAP82 and AKAP82 in mouse caput and cauda epididymidal spermatozoa. Immunofluorescence studies indicated that AKAP83 in hamster spermatozoa was localized along the length of principal piece of the tail. It was also demonstrated that hamster pro-AKAP83/AKAP83 gene expression was testis specific and was not expressed in other organs in either sex. This is the first report implicating AKAP in capacitation in rodents.
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PMID:Identification of the major tyrosine phosphorylated protein of capacitated hamster spermatozoa as a homologue of mammalian sperm a kinase anchoring protein. 1180 62


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