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)

Phosphorylations catalyzed by cAMP-dependent protein kinase are essential for sperm motility, and type II cAMP-dependent protein kinase in mature sperm has been shown to be firmly bound to the flagellum via the regulatory subunit, RII. The present study documents high-levelled expression of a human, testis-specific RII alpha mRNA (2.0 kb) analogous to the rat mRNA which is induced in haploid germ cells [(1988) FEBS Lett. 229, 391-394]. We report the molecular cloning of a full-length human cDNA corresponding to this unique testis mRNA, and the presence of an alternative amino-terminal region (amino acids 45-75) of the predicted RII alpha protein (404 amino acids) compared with the previously published mouse and rat sequences. However, this alternate region is also shown to be present in RII alpha mRNA (7.0 kb) of human somatic cells. Our data indicate the divergent amino-terminal sequence to be due to species differences, suggesting an active evolutionary pressure on this particular region, which could be involved in subcellular attachment of RII alpha and thereby localization of kinase activity to certain targets within the cell.
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PMID:Human testis cDNA for the regulatory subunit RII alpha of cAMP-dependent protein kinase encodes an alternate amino-terminal region. 254 40

The transcript for the high-affinity Ca2+/calmodulin-binding protein calspermin is generated from the gene encoding Ca2+/calmodulin-dependent protein kinase IV only in postmeiotic germ cells during spermatogenesis. We demonstrate that this testis-specific calspermin transcript can be produced in heterologous cells by utilization of a promoter located in an intron of the calmodulin (CaM) kinase IV gene. Critical motifs within this promoter are two cyclic AMP response element (CRE)-like sequences located about -70 and -50 bp upstream of the transcriptional initiation site. Both CRE motifs are footprinted by the authentic testis-specific transcriptional activator CREM tau or by CREM tau present in adult testis nuclear extract. Whereas a 2.1-kb DNA fragment containing the calspermin promoter is inactive when transfected into NIH 3T3 cells, activity can be restored by cotransfection of CREM tau and protein kinase A or CaM kinase IV but not CaM kinase II alpha. Restoration of activity is greatly reduced by mutation of the two CRE motifs. Since CRE-like motifs have been identified in many genes uniquely expressed in postmeiotic germ cells, which contain abundant CREM tau protein, we suggest that CREM tau may function as one transcription factor responsible for the expression of postmeiotic germ cell-specific genes.
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PMID:Calspermin gene transcription is regulated by two cyclic AMP response elements contained in an alternative promoter in the calmodulin kinase IV gene. 779 65

Transition protein 1 (TP1) is a small basic chromosomal protein that appears in mammalian spermatids during the period of chromatin condensation. The gene for TP1 from several species contains an apparent cAMP response element (CRE) in the immediate 5'-flanking region. The recent identification of high expression of the novel CRE-activating protein (CREM tau) in advanced testicular germ cells provided a stimulus to ask whether or not the TP1 CRE is functional. To this end we show both by gel retardation and by footprint assays that TP1 CRE forms specific bound complexes with proteins in whole testis nuclear extracts and that these complexes involve CREM as evidenced by recognition by a specific antibody. In addition, the TP1 CRE forms specific bound complexes with bacterially expressed CREM tau. Finally, the TPI CRE conveys protein kinase A-dependent induction to a linked chloramphenicol acetyl transferase gene when transfected into JEG-3 cells. Accordingly, TP1 is a good candidate for a testis-specific gene subject to CREM tau regulation.
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PMID:Identification of a functional cyclic adenosine 3',5'-monophosphate response element in the 5'-flanking region of the gene for transition protein 1 (TP1), a basic chromosomal protein of mammalian spermatids. 788 12

Using degenerate oligos corresponding to two highly conserved motifs within the protein kinase catalytic domain and a PCR-based cloning strategy, we have isolated a cDNA fragment encoding a new member of the Ser/Thr (serine/threonine) family of protein kinases. Expression analysis revealed that the fragment recognized two transcripts (1.6 and 1.4 kb) exclusively in testis. Using this fragment as a probe, we have cloned a full-length cDNA from a mouse testis cDNA library. The sequence has a 1092-bp open reading frame encoding a protein of 364 amino acids. The N-terminally localized kinase catalytic domain has all the conserved motifs found in other Ser/Thr kinases. Northern blot analysis using the full-length sequence as a probe revealed that the cloned gene corresponds to the 1.6-kb transcript, suggesting the existence of at least two testis-specific novel Ser/Thr kinases. We propose the name testis-specific kinase-1 (TSK-1) for the gene described here. A GenEMBL databank search revealed highest homology to the human gene encoding rac protein kinase-beta and the group of yeast Ser/Thr kinases encoded by SNF-1, nim-1, KIN-1 and KIN-2.
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PMID:Characterization of a novel murine testis-specific serine/threonine kinase. 811 11

Mammalian spermiogenesis is characterized by a striking restructuring of the spermatid chromatin caused by the replacement of nucleohistones with transition proteins and their subsequent replacement with nucleoprotamines. The onset of nuclear elongation and chromatin condensation in spermatids is accompanied by a general decrease in the transcriptional activity of the DNA. A recently identified testis-specific high-mobility-group (tsHMG) protein, similar to the human mitochondrial transcription factor I and to the linker-associated protein delta of Tetrahymena thermophila micronuclei, is thought to play a structural role in this process. We confirm by immunoblot analysis of fractionated germ cells that the presence of tsHMG is restricted to transcriptionally quiescent elongating and condensing spermatids. Purified recombinant tsHMG protein displays preferential binding to supercoiled plasmid DNA, which reversibly protects the DNA against the DNA-relaxing activity of eukaryotic topoisomerase I and also impairs the transcriptional activity of this template when assayed in vitro. The tsHMG protein can also introduce negative supercoils into a relaxed plasmid substrate in a topoisomerase I-dependent manner. We also show that the tsHMG protein is the substrate of a Ca2+-phospholipid-dependent protein kinase (protein kinase C) present in testis extracts of adult mice and demonstrate that phosphorylation by protein kinase C is required for both the DNA-binding and the topoisomerase I-dependent supercoiling activities of tsHMG. Our results support the hypothesis that the spermatid tsHMG protein is a topological factor (transition protein) that can modulate the activity of topoisomerase I. This activity could contribute to the important transition in chromatin structure which leads to the decrease in DNA metabolism observed at the early stages of spermatid elongation.
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PMID:The testis-specific high-mobility-group protein, a phosphorylation-dependent DNA-packaging factor of elongating and condensing spermatids. 866 89

Immotile bovine caput epididymal sperm contain levels of protein phosphatase activity twofold higher than do mature motile caudal sperm. Comparison of the inhibition profiles of endogenous phosphatase activities detected by okadaic acid (OA) and calyculin A (CA) revealed a pattern consistent with the predominance of a type 1 protein phosphatase (PP1). Immunoblot analysis identified PP1 gamma 2 (the testis-specific isoform of PP1) as the only PP1 isoform in sperm and showed little protein phosphatase 2A (PP2A). In addition, of the known PP1 inhibitors, i.e., DARPP-32, inhibitor 1 (I1), and inhibitor 2 (I2), only I2-like activity was detected in sperm. Inhibition of PP1 by the heat-stable I2-like activity purified from sperm could be reversed with purified glycogen synthase kinase-3 (GSK-3). Furthermore, sperm extracts contain an inactive complex of PP1 and I2 (termed PP1I) that could also be activated by purified GSK-3. The presence of GSK-3 in sperm was demonstrated by activation of purified PP1I, and quantitation revealed that immotile caput sperm contained sixfold higher GSK-3 activity than motile caudal sperm. Immunoblot analysis confirmed the expression of GSK-3 in sperm and revealed the occurrence of both the alpha and beta isoforms. Our findings suggest that the higher PP1 activity measured in immotile sperm, presumably due to higher GSK-3 activity, is responsible for holding motility in check. This conclusion was supported by the observation that the phosphatase inhibitors OA and CA, at micromolar and nanomolar levels, respectively, were able to induce motility in completely immotile bovine caput epididymal sperm and to stimulate the kinetic activity of mature caudal sperm. The intrasperm levels of cAMP, pH, and calcium were unaltered by treatment with these inhibitors. The results suggest a biochemical basis for the development and regulation of sperm motility and a possible physiological role for the PP1/I2/GSK-3 system.
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PMID:Sperm motility development in the epididymis is associated with decreased glycogen synthase kinase-3 and protein phosphatase 1 activity. 883 95

The acquisition of autosomal fertility genes has been proposed to be an important process in human Y chromosome evolution. For example, the Y-linked fertility factor DAZ (Deleted in Azoospermia) appears to have arisen after the transposition and tandem amplification of the autosomal DAZH gene. The Drosophila melanogaster Y chromosome contains tandemly repeated Su(Ste) units that are thought to affect male fertility as suppressors of the homologous X-linked Stellate repeats. Here we report the detection of a testis-expressed autosomal gene, SSL [Su(Ste)-like], that appears to be an ancestor of the Y-linked Su(Ste) units. SSL encodes a casein kinase 2 (CK2) beta-subunit-like protein. Its putative ORF shares extensive (45%) homology with the genuine beta-subunit of CK2 and retains the conserved C-terminal and Glu/Asp-rich domains that are essential for CK2 holoenzyme regulation. SSL maps within region 60D1-2 of D. melanogaster and D. simulans polytene chromosomes. We present evidence that SSL was derived from the genuine betaCK2 gene by reverse transcription. This event resulted in the loss of the first three introns in the coding region of the SSL ancestor gene. Evolutionary analysis indicates that SSL has evolved under selective pressure at the translational level. Its sequence, especially in the 3' region, is much closer to the Y-linked Su(Ste) tandem repeats than to the betaCK2 gene. These results suggest that the acquisition of testis-specific autosomal genes may be important for the evolution of Drosophila as well as human Y chromosomes.
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PMID:Acquisition and amplification of a testis-expressed autosomal gene, SSL, by the Drosophila Y chromosome. 917 11

cAMP is important for the initiation of mammalian sperm motility. Previously we established that a type II cAMP-dependent protein kinase is tightly associated with the fibrous sheath of rat sperm. This unique cytoskeletal structure surrounds the 9+2 axonemal network in the principal piece of the flagellum. Association of the kinase to the fibrous sheath is mediated via its regulatory subunit, RII. An RII-binding overlay procedure was used to document that RII could specifically associate with fibrous sheath polypeptides of 120 and 80 kDa. In this study, we report the cloning of a rat testis-specific, developmentally regulated, RII-binding protein (TAKAP-80). A 1.2-kb cDNA clone, isolated by screening a rat testis expression library with 32P-labeled RII, hybridized to a 1.8-kb mRNA transcript present exclusively in testis. This transcript appeared at detectable levels at 30 days after birth. Over the next 10 days the mRNA levels increased greatly. This time interval corresponds to the initiation of spermiogenesis. The complete nucleotide sequence of TAKAP-80 cDNA was obtained by polymerase chain reaction and contained a continuous open reading frame of 502 amino acids. The deduced amino acid sequence showed a clear demarcation of charged and hydrophobic amino acid residues. Amino acids 1-147 of the protein contained 45% charged residues, with lysine and arginine predominating. Similarly, amino acids 268-502 also contained a high percentage of charged amino acids (35%). In contrast, amino acids 148-267 were mostly hydrophobic and contained clusters of a repeating PXXP motif where X was predominantly valine and alanine or sometimes proline. The 1.2-kb cDNA clone was inserted into the pRSET vector and expressed as a His6 tag fusion protein in Escherichia coli. The recombinant protein was soluble and bound RIIalpha, RIIbeta and type IIalpha holoenzyme by the RII-binding overlay procedure. Deletion analysis revealed that the high-affinity interaction site for RII was contained within amino acids 258-378 of TAKAP-80. Antibodies prepared against the fusion protein recognized an 80-kDa protein present in the urea-insoluble particulate fraction of rat testis and in purified fibrous sheath preparations isolated from rat epididymal sperm. Levels of the 80-kDa immunoreactive protein were significantly higher in mature (60 days old) compared with immature (30 days old) rat testis, correlating with the mRNA levels.
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PMID:Cloning and characterization of a testis-specific, developmentally regulated A-kinase-anchoring protein (TAKAP-80) present on the fibrous sheath of rat sperm. 920 34

The molecular mechanisms regulating the spectacular cytodifferentiation observed during spermiogenesis are poorly understood. We have recently identified a murine testis-specific serine kinase (tssk) 1, constituting a novel subfamily of serine/threonine kinases. Using low stringency screening we have isolated and molecularly characterized a second closely related family member, tssk 2, which is probably the orthologue of the human DGS-G gene. Expression of tssk 1 and tssk 2 was limited to the testis of sexually mature males. Immunohistochemical staining localized both kinases to the cytoplasm of late spermatids and to structures resembling residual bodies. tssk 1 and tssk 2 were absent in released sperms in the lumen of the seminiferous tubules and the epididymis, demonstrating a tight window of expression restricted to the last stages of spermatid maturation. In vitro kinase assays of immunoprecipitates containing either tssk 1 or tssk 2 revealed no autophosphorylation of the kinases, however, they led to serine phosphorylation of a coprecipitating protein of approximately 65 kD. A search for interacting proteins using the yeast two-hybrid system with tssk 1 and tssk 2 cDNA as baits and a prey cDNA library from mouse testis, led to the isolation of a novel cDNA, interacting specifically with both tssk 1 and tssk 2, and encoding the coprecipitated 65-kD protein phosphorylated by both kinases. Interestingly, expression of the interacting clone was also testis specific and paralleled the developmental expression observed for the kinases themselves. These results represent the first demonstration of the involvement of a distinct kinase family, the tssk serine/threonine kinases, together with a substrate in the cytodifferentiation of late spermatids to sperms.
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PMID:A novel family of serine/threonine kinases participating in spermiogenesis. 941 77

Three different catalytic isoforms of cAMP-dependent protein kinase have been identified (C alpha, C beta, and C gamma). We report the cloning and characterization of the human and rhesus monkey genes encoding the testis-specific C gamma subunit. The human C gamma gene is intronless with an open reading frame similar to the previously published cDNA sequence. The 3' and 5' flanking regions share high similarity with the C alpha nontranslated regions (82%) also outside the regions corresponding to the C gamma cDNA. The human gene is flanked by an Alu-related sequence in the 5'-end and there are insertions of two Alu-related sequences in the 3' nontranslated region. The observation that the C gamma gene is intronless and colinear with C alpha mRNA, together with the presence of remnants of a poly(A) tail and flanking direct repeats, indicates that the C gamma gene is a C alpha-derived retroposon. The 5' flanking region of this gene has a high G/C content and a putative TATA box situated at -138 compared to the translation initiation codon. Cloning and sequencing of a partial C gamma rhesus monkey gene demonstrate conservation of the sequence in primates. Northern analysis on isolated and fractionated human germ cells of testes from normal and estrogen-treated individuals demonstrates that the C gamma gene is expressed only in germ cells in the human testis. Our results indicate that the C gamma gene is a retroposon specifically transcribed in primate testicular germ cells.
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PMID:The gene encoding the C gamma catalytic subunit of cAMP-dependent protein kinase is a transcribed retroposon. 959 17


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