Tore Jahnsen

Cloning and characterization of a cDNA encoding an A‐kinase anchoring protein located in the centrosome, AKAP450

A combination of protein kinase A type II (RII) overlay screening, database searches and PCR was used to identify a centrosomal A‐kinase anchoring protein. A cDNA with an 11.7 kb open reading frame was characterized and found to correspond to 50 exons of genomic sequence on human chromosome 7q21‐22. This cDNA clone encoded a 3908 amino acid protein of 453 kDa, that was designated AKAP450 (DDBJ/EMBL/GenBank accession No. AJ131693). Sequence comparison demonstrated that the open reading frame contained a previously characterized cDNA encoding Yotiao, as well as the human homologue of AKAP120. Numerous coiled‐coil structures were predicted from AKAP450, and weak homology to pericentrin, giantin and other structural proteins was observed. A putative RII‐binding site was identified involving amino acid 2556 of AKAP450 by mutation analysis combined with RII overlay and an amphipatic helix was predicted in this region. Immunoprecipitation of RII from RIPA‐buffer extracts of HeLa cells demonstrated co‐precipitation of AKAP450. By immunofluorecent labeling with specific antibodies it was demonstrated that AKAP450 localized to centrosomes. Furthermore, AKAP450 was shown to co‐purify in centrosomal preparations. The observation of two mRNAs and several splice products suggests additional functions for the AKAP450 gene.

Molecular cloning and predicted full‐length amino acid sequence of the type Iβ isozyme of cGMP‐dependent protein kinase from human placenta

In this study we report the isolation and characterization of three overlapping cDNA clones for the type Iβ isozyme of cGMP‐dependent protein kinase (cGK) from human placenta libraries. The composite sequence was 3740 nucleotides long and contained 58 nucleotides from the 5′‐noncoding region, an open reading frame of 2061 bases including the stop codon, and a 3′‐noncoding region of 1621 nucleotides. The predicted full‐length human type Iβ cGK protein contained 686 amino acids including the initiator methionine, and had an estimated molecular mass of 77 803 Da. On comparison to the published amino acid sequence of bovine lung Iα, human placenta Iβ cGK differed by only two amino acids in the carboxyl‐terminal region (amino acids 105–686). In contrast, the amino‐terminal region of the two proteins was markedly different (only 36% similarity), and human Iβ cGK was 16 amino acids longer. In a specific region in the amino‐terminus (amino acids 63–75), 12 out of 13 amino acids of the human Iβ cGK were identical to the partial amino acid sequence recently published for a new Iβ isoform of cGK from bovine aorta. Northern blot analysis demonstrated a human Iβ cGK mRNA, 7 kb in size, in human uterus and weakly in placenta. An mRNA of 7 kb was also observed in rat cerebellum, cerebrum, lung, kidney, and adrenal, whereas an mRNA doublet of 7.5 and 6.5 kb were observed in rat heart. Comparison of Northern and Western blot analyses demonstrated that the mRNA and protein for cerebellar cGK increased during the development of rats from 5 to 30 days old, whereas the 6.5 kb mRNA in rat heart declined.

Human testis cDNA for the regulatory subunit RIIα of CAMP-dependent protein kinase encodes an alternate amino-terminal region

Phosphorylations catalysed 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 RIIα 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 alternate amino‐terminal region (amino acids 45–75) of the predicted RIIα protein (404 amino acids) compared with the previously published mouse and rat sequences. However, this alternate region is also shown to be present in RIIα 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 RIIα and thereby localization of kinase activity to certain targets within the cell.

Molecular cloning of the human transmembrane secretory component (poly-Ig receptor) and its mRNA expression in human tissues.

A 2.5 kilobase (kb) cDNA clone containing 92% of the coding region for human transmembrane secretory component (SC) or poly-Ig receptor, was isolated from a mammary gland cDNA library. The cDNA clone encoded a protein of 693 amino acids which showed 99% homology with the primary amino acid sequence of human free SC as reported by Eiffert et al. (1), and 54% homology with the deduced amino acid sequence of rabbit transmembrane SC for which cDNA was cloned by Mostov et al. (2). Northern blot analysis showed mRNA expression in various human exocrine tissues in good agreement with our previous immunohistochemical studies of SC.

Molecular cloning, cDNA structure and deduced amino acid sequence for a type I regulatory subunit of cAMP-dependent protein kinase from human testis

A 1.5 kilobase (kb) cDNA clone containing the entire coding region for a regulatory subunit of type I cAMP-dependent protein kinase (RI) was isolated from a human testis cDNA library. The cDNA clone encodes a protein of 381 amino acids that shows 98% and 97% homology to the bovine skeletal muscle RI and rat brain RI, respectively. Northern blot analysis demonstrates two major mRNA-species (1.5 and 3.0 kb) in human testis and one mRNA-species (3.0 kb) in human T-lymphocytes.

Molecular cloning, cDNA structure and tissue-specific expression of the human regulatory subunit RIβ of cAMP-dependent protein kinases

Abstract Complementary DNA clones for the regulatory subunit RIβ of cAMP-dependent protein kinases were isolated from a human testis cDNA library using a mouse RIβ cDNA probe. One clone 2.4 kilobases (kb) in length contained an open reading frame of 1137 bases, and encoded a protein of 379 amino acids (excluding the initiator methionine). The human RIβ protein was one amino acid shorter than the corresponding protein in mouse and rat. The nucleotide similarity to mouse and rat sequences was 85.6% and 84.8%, respectively, while the amino acid similarity was 97.6% and 97.3%, respectively. Northern blot analyses revealed a 2.7 kb mRNA in human tissues and a 2.8 kb mRNA in mouse tissues. Both mouse and human RIβ mRNA were found to be expressed in most tissues, and not restricted to brain and testis as reported by others.

Amplification and protein over-expression of the neu /HER-2/c- erbB -2 protooncogene in human breast carcinomas: relationship to loss of gene sequences on chromosome 17, family history and prognosis

c-erbB-2 gene amplification and protein over-expression were investigated in 89 primary tumours and 24 metastases from Norwegian breast cancer patients. Amplification occurred in 22.5% of the primary tumours and 50% of the metastases. The amplification was negatively correlated to the oestrogen receptor (ER) content in both the primary tumours and the metastases. No significant differences between amplified and non-amplified tumours were observed with regard to node status, clinical stage, tumour size or menopausal status, although correlations of borderline significance were found between node status, clinical stage and high degree of gene amplification. All the amplified tumours were of the invasive ductal type. Follow-up data of patients observed for more than 1 year showed a significantly higher recurrence rate in the c-erbB-2 amplified group. Allele loss of chromosome 17p and of 7q was seen in 55% and 48% of the tumours respectively. No significant correlation was found between these losses and clinico-histological parameters. More than 50% of the tumours with a loss of 17q sequences had an amplification of c-erbB-2 which is located on 17q12-21, indicating that only one of the chromosomes may be involved in the amplification of the c-erbB-2. A trend towards a correlation between loss of 17q and high degree of amplification were found. No correlation was found between positive family history of breast cancer and c-erbB-2 gene amplification, nor loss of 17p or 17q sequences. Our data support the hypothesis that amplification correlates with aggressive tumour behaviour, and thus may be used as a prognostic factor in breast carcinomas. The allele losses on 17p and 17q points to tumour suppressor gene or genes on this chromosome, although not as predisposing genes in families.

FAST AND SLOW CYCLIC NUCLEOTIDE-DISSOCIATION SITES IN CAMP-DEPENDENT PROTEIN KINASE ARE TRANSPOSED IN TYPE IBETA CGMP-DEPENDENT PROTEIN KINASE

Both cyclic GMP-dependent protein kinase (cGK) and cyclic AMP-dependent protein kinase (cAK) contain two distinct cyclic nucleotide-binding sites referred to as fast and slow sites based on cyclic nucleotide dissociation behavior. In cAK, the fast site lies amino-terminal to the slow site, and sequence homologies between cAK and cGK have suggested similar positioning for the sites in cGK. Recombinant human type Iβ cGK (wild type (WT) cGK) was overexpressed, and the properties of purified WT cGK and native type Iβ cGK were similar. cGK was mutated singly at Thr-193 (T193A, T193V, and T193S) and Thr-317 (T317A, T317V, and T317S), which have been predicted to provide cGMP specificity in the cGMP-binding sites of cGK; a double mutant (T193A/T317A) was produced also. Compared with WT cGK, half-maximal activation (Ka) of mutant cGKs by cGMP was increased 2- (T317A), 27- (T193A), or 63-fold (T193A/T317A), but the Ka for cAMP of these mutants was essentially unchanged. The T193A and T193V mutants had a large increase in the rate of the slow component of [3H]cGMP dissociation, but in the T317A and T317V mutants, there was no change in the slow component. The T193S and T317S mutants had only minor effects on [3H]cGMP dissociation, thus establishing the importance of the hydroxyl group of Thr-193 and −317 for cGMP binding to cGK. Thus, in type Iβ cGK, the slow cGMP-binding site is identified as the amino-terminal site in contrast to the order assigned to the fast and slow cAMP-binding sites of cAK.

Exogenous pyruvate accelerates glycolysis and promotes capacitation in human spermatozoa

BACKGROUND There has been an ongoing debate in the reproductive field about whether mammalian spermatozoa rely on glycolysis, oxidative phosphorylation or both for their energy production. Recent studies have proposed that human spermatozoa depend mainly on glucose for motility and fertilization but the mechanism behind an efficient glycolysis in human spermatozoa is not well understood. Here, we demonstrate how human spermatozoa utilize exogenous pyruvate to enhance glycolytic ATP production, motility, hyperactivation and capacitation, events that are crucial for male fertility. METHODS Purified human spermatozoa from healthy donors were incubated under capacitating conditions (including albumin, bicarbonate and glucose) and tested for changes in ATP levels, motility, hyperactivation and tyrosine phosphorylation after treatment with pyruvate. The experiments were repeated in the presence of sodium cyanide in order to assess the contribution from mitochondrial respiration. The metabolism of 13C labeled glucose and pyruvate was traced by a combination of liquid chromatography and mass spectrometry. RESULTS The treatment of human spermatozoa with exogenous pyruvate increased intracellular ATP levels, progressive motility and hyperactivation by 56, 21 and 130%, respectively. In addition, added pyruvate induced a significant increase in tyrosine phosphorylation levels. Blocking of the electron transport chain did not markedly affect the results, indicating that the mechanism is independent of oxidative phosphorylation. However, the observed effects could be counteracted by oxamate, an inhibitor of lactate dehydrogenase (LDH). Metabolic tracing experiments revealed that the observed rise in ATP concentration resulted from an enhanced glycolytic flux, which was increased by more than 50% in the presence of exogenous pyruvate. Moreover, all consumed 13C labeled pyruvate added was converted to lactate rather than oxidized in the tricarboxylic acid cycle. CONCLUSIONS Human spermatozoa seem to rely mainly, if not entirely, on glycolysis as the source of ATP fueling the energy-demanding processes of motility and capacitation. The efficient glycolysis is dependent on exogenous pyruvate, which indirectly feeds the accelerated glycolysis with NAD+ through the LDH-mediated conversion of pyruvate to lactate. Pyruvate is present in the human female reproductive tract at concentrations in accordance with our results. As seen in other mammals, the motility and fertility of human spermatozoa seem to be dictated by the available energy substrates present in the conspecific female.

A Novel Isoform of Human Cyclic 3′,5′-Adenosine Monophosphate-Dependent Protein Kinase, Cα-s, Localizes to Sperm Midpiece

Abstract Using rapid amplification of cDNA ends, a cDNA encoding a novel splice variant of the human Cα catalytic subunit of cAMP-dependent protein kinase (PKA) was identified. The novel isoform differed only in the N-terminal part of the deduced amino acid sequence, corresponding to the part encoded by exon 1 in the previously characterized murine Cα gene. Sequence comparison revealed similarity to an ovine Cα variant characterized by protein purification and micropeptide sequencing, Cα-s, identifying the cloned human cDNA as the Cα-s isoform. The Cα-s mRNA was expressed exclusively in human testis and expression in isolated human pachytene spermatocytes was demonstrated. The Cα-s protein was present in ejaculated human sperm, and immunofluorescent labeling with a Cα-s-specific antibody indicated that Cα-s was localized in the midpiece region of the spermatozoon. The majority of Cα-s was particulate and could not be released from the sperm midpiece by cAMP treatment alone. Furthermore, detergent extraction solubilized approximately two-thirds of the Cα-s pool, indicating interaction both with detergent-resistant cytoskeletal and membrane structures. In addition, we recently identified the regulatory subunit isoforms RIα, RIIα, and an A-kinase anchoring protein, hAKAP220 in this region in sperm that could target Cα-s. This novel Cα-s splice variant appeared to have an independent anchor in the human sperm midpiece as it could not be completely solubilized even in the presence of both detergent and cAMP.