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V Hansson

Publications and source records attributed to V Hansson.

At least 37 records · Page 2Linked to original sources
PubMed1997

Molecular cloning, upstream sequence and promoter studies of the human gene for the regulatory subunit RII alpha of cAMP-dependent protein kinase.

The gene for the regulatory subunit RII alpha of cAMP-dependent protein kinase is highly regulated during spermatogenesis and a strong signal from a distinct short mRNA form is observed postmeiotically during spermatid elongation. This report presents the isolation and characterization of the 5'-flanking region (1.2 kb) and exon 1 of the human RII alpha gene. S1 nuclease mapping and primer extension experiments revealed the presence of a major transcriptional start site located 208 nucleotides upstream of start for translation. The 5'-flanking region of the RII alpha gene did not contain a TATA box and was highly G/C-rich. A basal promoter directing high levels of chloramphenicol acetyl transferase (CAT) activity was identified in the 5'-flanking sequence. Several potential binding sites for transcription factors were identified in this region, which may be responsible for the germ cell-specific regulation of this gene. We have previously reported that the human testis RII alpha cDNA contains a region (amino acids 45-75) with little or no homology to the corresponding rat skeletal muscle cDNA (Oyen, O., Myklebust, F., Scott, J.D., Cadd, G.G., McKnight, G.S., Hansson, V. and Jahnsen, T. (1990) Biol. Reprod. 43, 46-54). We examined whether this difference could arise due to organ-specific splice mechanisms or represented a species difference. We show that the low homology region of the human RII alpha cDNA resides entirely within exon 1, and does not originate from a tissue-specific alternate splicing of this distinct region.

Amino Acid Sequence↗
PubMed1997

Structure, function, and regulation of human cAMP-dependent protein kinases.

A large number of hormones, neurotransmitters, and other signaling substances that bind to G-protein-coupled cell-surface receptors have their signals converge at one sole second messenger, cAMP. The question of how specificity can be maintained in a signal-transduction system in which many extracellular signals leading to a vast array of intracellular responses are all mediated through one second-messenger system has been the subject of thorough investigation and a great deal of speculation. An increasing number of cAK isozymes, consisting of homo- or heterodimers of R subunits (RIalpha, RIbeta, RIIalpha, RIIbeta) with associated catalytic subunits (C alpha, Cbeta, Cgamma), may, at least in part, explain this specificity. The various cAK isozymes display distinct biochemical properties, and the heterogeneous subunits of cAK reveal cell-specific expression and differential regulation at the level of gene transcription, mRNA stability, and protein stability in response to a wide range of hormones and other signaling substances. The existence of a number of anchoring proteins specific to either RIIalpha or RIIbeta, and which localize cAKII isozymes toward distinct substrates at defined subcellular loci, strongly supports the idea that specific functions can be assigned to the various cAK isozymes. The demonstration that selective activation of cAKI is necessary and sufficient for cAMP-mediated inhibition of T-cell proliferation, and the observation that T-cell activation is associated with redistribution and colocalization of cAKI to the TCR, is also compatible with the notion of isozyme-specific effects.

Cloning, Molecular↗
PubMed1997

The human gene for the regulatory subunit RI alpha of cyclic adenosine 3', 5'-monophosphate-dependent protein kinase: two distinct promoters provide differential regulation of alternately spliced messenger ribonucleic acids.

The present study reports the exon-intron organization of the human RI alpha gene of cAMP-dependent protein kinase and approximately kilobases (kb) of the 5'-flanking region obtained by isolation and sequencing of several phage clones from human genomic libraries. The RI alpha gene is composed of nine coding exons of varying lengths, separated by introns, giving the gene a total length of at least 21 kb. our recent cloning of a processed RI alpha pseudogene with a 5'-noncoding region different from the previously reported RI alpha complementary RNA indicated that the RI alpha gene may have multiple leader exons giving rise to alternately spliced messenger RNAs (mRNAs). Reverse transcription of human testis RNA followed by PCR identified two different RI alpha mRNA species (RI alpha 1a and RI alpha 1b) containing distinct sequences due to alternately splicing the gene. The previously known RI alpha 1b mRNA revealed low constitutive expression in a human B lymphoid cell line (Reh) and was stimulated only 4- to 6-fold by treatment with cAMP. In contrast, very low levels of the novel RI alpha 1a mRNA were present in untreated Reh cells, but were stimulated 40-to 50-fold by cAMP. The 5'-flanking sequence of the RI alpha gene was G/C rich and did not contain any TATA box. Several putative transcription initiation sites were identified in front of each leader exon (exons 1a and 1b) by the 5'-rapid amplification of complementary DNA ends technique. To determine whether the sequences 5' of both leader exons had promoter activities, the 5'-flanking sequences of exons 1a and 1b were inserted in front of a chloramphenicol acetyltransferase reporter gene, and their ability to direct transcription were examined. Transfection of these constructs into rat GH4C1 cells demonstrated that both constructs had promoter activities, as evidenced by high levels of chloramphenicol acetyltransferase activity.

Aged↗
PubMed1996

The gene encoding the catalytic subunit C alpha of cAMP-dependent protein kinase (locus PRKACA) localizes to human chromosome region 19p13.1.

We have determined the chromosomal localization of the gene for the catalytic subunit C alpha of cAMP-dependent protein kinase (locus PRKACA) to human chromosome 19 using polymerase chain reaction (PCR) and Southern blot analysis of two different somatic cell hybrid mapping panels. In addition, PCR analysis of a chromosome 19 mapping panel revealed the presence of a human C alpha-specific amplification product only in cell lines containing the region 19p13.1 to 19q12. Finally, two-color fluorescence in situ hybridization to metaphase chromosomes using the human C alpha cDNA and human chromosome 19 inter-Alu-PCR product as probes localized the human C alpha gene to chromosome region 19p13.1.

Animals↗
PubMed1996

Molecular cloning, cDNA structure, and chromosomal localization of the human type II cGMP-dependent protein kinase.

The type II cGMP-dependent protein kinase is an enzyme originally isolated from the small intestine, and is thought to be involved in the regulation of intestinal ion transport and fluid secretion. A complementary DNA clone encoding a part of the human type II cGMP-dependent protein kinase was isolated from a cerebellum library. Based on sequence information from this complementary DNA, the 5'-end of the type II cGMP-dependent protein kinase was amplified from human brain messenger RNA using polymerase chain reaction. The composite complementary DNA encoded a 762 amino acid protein with a calculated molecular mass of 87.4 kDa. Messenger RNAs encoding the type II cGMP-dependent protein kinase were detected in small intestine, colon and prostate. By using polymerase chain reaction and Southern blotting on somatic cell hybrids, the gene encoding, the type II cGMP-dependent protein kinase was mapped to human chromosome 4q13.1-q21.1.

Amino Acid Sequence↗
PubMed1996

Cyclic AMP-dependent protein kinase (cAK) in human B cells: co-localization of type I cAK (RI alpha 2 C2) with the antigen receptor during anti-immunoglobulin-induced B cell activation.

Cyclic AMP (cAMP) inhibits antigen-stimulated B cell proliferation through activation of cAMP-dependent protein kinases (cAK). We have examined the molecular composition and cellular localization of cAK in human B cells. We find that human B cells contain substantial amounts of mRNA for RI alpha, RII alpha, C alpha and C beta, barely detectable levels of RI beta mRNA, and no detectable RII beta or C gamma mRNA. At the protein level, using Western blotting and subunit-specific antibodies against the different R subunits, we find RI alpha and RII alpha, but no RI beta or RII beta. The presence of catalytic subunits was demonstrated using a nonselective anti-C antiserum. By photoaffinity labeling of R subunits with 8-azido-[32P]cAMP, followed by immunoprecipitation with subunit-specific antibodies, we were also able to demonstrate low levels of RI beta. Immunofluorescence staining of RI alpha and RII alpha demonstrates a rather homogeneous intracellular (but extranuclear) distribution of RI alpha, whereas the RII alpha subunits of cAK are localized to distinct perinuclear structures, previously identified as centrosomes in other cell types. Upon anti-Ig-mediated capping of B cells, RI alpha subunits redistribute to the cap, co-localizing with the antigen-receptors, whereas the intracellular localization of RII alpha subunits remains unchanged.

B-Lymphocytes↗
PubMed1996

Regulation of protein kinase A subunits by cyclic adenosine 3',5'-monophosphate in a mouse Sertoli cell line (MSC-1): induction of RII beta messenger ribonucleic acid is independent of continuous protein synthesis.

We report the basal and cAMP-regulated expression of protein kinase A (PKA) subunits in a mouse Sertoli cell line (MSC-1). Of the PKA subunits expressed by these cells (RI alpha, RII alpha, RII beta, C alpha, C beta), only RII beta was regulated by cAMP. An approximately 8-fold induction of RII beta mRNA and a 3-fold induction of RII beta protein was observed during 48 h of cAMP-stimulation. This cAMP-mediated RII beta mRNA induction, reaching maximal levels after approximately 12 h, did not require ongoing protein synthesis. Fairly rapid decay of maximally induced RII beta mRNA was observed after removal of cAMP (t1/2 approximately 5 h). Further, ongoing transcription and translation were necessary for rapid degradation of RII beta mRNA. Thus, the MSC-1 cells expressed all the PKA subunits present in primary cultures of Sertoli cells and responded to cAMP with increased levels of RII beta at both mRNA and protein levels. Although the nature of some of these responses distinguished the observations in MSC-1 cells from previously described responses in primary cultures, these cells may prove to be useful in future studies addressing cAMP-mediated gene regulation.

Animals↗
PubMed1995

The alpha-subunit mRNAs for Gs and Go2 are differentially regulated by protein kinase A and protein kinase C in rat Sertoli cells.

In the present study, we have examined regulatory effects of protein kinase A and protein kinase C activation by 8-CPTcAMP and TPA, respectively, on mRNAs for various G protein alpha-subunits and corresponding immunoreactive proteins in rat Sertoli cells. Gs alpha and Go alpha mRNA levels were transiently increased 1.5-fold and 4-fold, respectively, by 8-CPTcAMP in cultured Sertoli cells. This up-regulation of mRNAs for Gs alpha and Go alpha was also observed when Sertoli cells were incubated in the presence of FSH. When protein synthesis was inhibited by cycloheximide, the cAMP-mediated stimulation of Gs alpha mRNA was abolished, whereas Go alpha mRNA was superinduced to a 50- to 100-fold higher level than basal. Activation of protein kinase C with TPA had a strong, synergistic effect on cAMP-mediated stimulation of Gs alpha mRNA, whereas the cAMP-mediated stimulation of Go alpha mRNA was completely blocked. Surprisingly, changes in mRNA levels were not accompanied by any alterations in the levels of immunoreactive Gs alpha and Go alpha proteins.

Animals↗
PubMed1995

Modulation of gonadotropic and purinergic responsiveness by islet activating protein in sertoli cells from immature rats.

In the present study we have examined the effects of islet activating protein (IAP) on the regulatory effects of FSH, glucagon and (-)N6-(R)-phenyl-isopropyladenosine (PIA), an adenosine A1 receptor agonist, on the formation of cAMP and estradiol-17 beta (E2) in Sertoli cell cultures isolated from immature (19-day-old) rats. FSH (NIH-FSH-S-15) (1.25 micrograms/ml) caused a more than 10-fold stimulation of the level of both cAMP and E2 in the spent media from Sertoli cell cultures during an 18 h incubation. Both responses were reduced by 80% in the presence of PIA (10(-6) M). When the cultures were preincubated for 24 h with increasing concentration of IAP, the inhibitory effects of PIA were counteracted in a concentration-dependent manner. Moreover, preincubation with IAP (> 20 ng/ml) caused a significant stimulation of FSH-stimulated cAMP production even in the absence of PIA. PIA inhibited FSH-stimulated cAMP production in a concentration dependent manner. However, when the cells were preincubated with IAP (100 ng/ml) for 24 h, the inhibitory effects of PIA were completely abolished, and PIA now actually caused a slight stimulation of cAMP production. Both FSH and glucagon stimulated cAMP production in a concentration-dependent manner. Preincubation with IAP (100 ng/ml for 24 h) resulted in an increase in maximal stimulation of cAMP production for both FSH and glucagon. When adenylyl cyclase (AC) activity was measured directly in isolated membrane particles from Sertoli cells cultured in the presence of IAP (100 ng/ml) for 24 h, both basal and FSH-stimulated AC activity were significantly higher than in membrane particles from control cells. These results provide a further characterization of the functional Gi component coupled to the AC complex in cultured rat Sertoli cells, mediating the inhibitory effects of adenosine and possibly other endogenous substances on cAMP production.

Adenosine↗
PubMed1994

Characterization of in-vitro-translated human regulatory and catalytic subunits of cAMP-dependent protein kinases.

Full-length human cDNAs for all the different regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinases (PKA) were transcribed and translated in a cell-free in vitro system. The resulting proteins were characterized with respect to molecular size, isoelectric focusing, immunoreactivity, cAMP binding, and to what extent the RII protein subunits revealed mobility shifts upon phosphorylation by catalytic subunit of PKA. We were able to express cDNAs for all the human R (RI alpha, RI beta, RII alpha and RII beta) and C (C alpha, C beta and C gamma) subunits in a wheat-germ extract. [35S]Methionine-labelled in-vitro-translated products were analyzed by SDS/PAGE and revealed distinct protein bands with apparent molecular masses of 49 (RI alpha), 54-55 (RI beta), 51 (RII alpha) and 53 kDa (RII beta) for the R subunits. In vitro transcription/translation of the cDNAs for the C subunits of PKA gave proteins with molecular masses of approximately 40 kDa for all the different C subunits. Phosphorylation of RII alpha and RII beta by the C subunit of PKA, revealed a distinct mobility shift of the RII alpha subunit on one-dimensional SDS/PAGE (51-54 kDa), but not of RII beta (53 kDa). Further characterization of the R subunits by two-dimensional SDS/PAGE revealed that RI alpha was more acidic than RI beta, with pIs of 6.1-6.0 and 6.4-6.2, respectively. Furthermore, the RII alpha protein was more basic than RII beta, with pIs of approximately 5.4-5.3 and 5.3-5.1, respectively. All the in-vitro-translated R subunits could be photoaffinity labelled by the cAMP-analog 8-azido-[32P]cAMP and were also detected by immunoprecipitation with subunit-specific antibodies.

Affinity Labels↗
PubMed1994

Location of cAMP-dependent protein kinase type I with the TCR-CD3 complex.

Selective activation of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase type I (cAKI), but not type II, is sufficient to mediate inhibition of T cell replication induced through the antigen-specific T cell receptor-CD3 (TCR-CD3) complex. Immunocytochemistry and immunoprecipitation studies of the molecular mechanism by which cAKI inhibits TCR-CD3-dependent T cell replication demonstrated that regulatory subunit I alpha, along with its associated kinase activity, translocated to and interacted with the TCR-CD3 complex during T cell activation and capping. Regulatory subunit II alpha did not. When stimulated by cAMP, the cAKI localized to the TCR-CD3 complex may release kinase activity that, through phosphorylation, might uncouple the TCR-CD3 complex from intracellular signaling systems.

Carrier Proteins↗
PubMed1994

The mature form of interleukin-1 alpha is constitutively expressed in immature male germ cells from rat.

In the present study we show that immature germ cells from rat testis contain both interleukin-1 alpha (IL-1 alpha) mRNA and immunoreactive proteins. In contrast, in primary cultures of Sertoli cells and peritubular cells. IL-1 alpha mRNA and immunoreactive protein were below the levels of detection. Immunoblots of lysates from these germ cells showed the presence of strong 17 kDa bands (mature forms of IL-1 alpha) and a much weaker 33 kDa band (precursor form). The finding of cell-associated mature forms of IL-1 alpha in germ cells indicates that immature male germ cells are able to process IL-1 alpha independent of its secretion. Data from isolated cell fractions, as well as from whole testis tissue from rats of various ages, indicate that IL-1 alpha expression takes place in late pachytene spermatocytes and early round spermatids. Whether IL-1 alpha plays a role intracellularly in germ cells or exerts its effects on neighboring Sertoli cells remains to be shown.

Animals↗
PubMed1994

Regulation of glucocorticoid receptor (GR) mRNA and protein levels by phorbol ester in MCF-7 cells. Mechanism of GR mRNA induction and decay.

Treatment of MCF-7 cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (10(-7) M) was associated with a time-dependent increase in specific binding of [3H]dexamethasone (34.8 +/- 4.6 fmol/mg protein after 9 h of TPA treatment compared with 16.0 +/- 2.3 fmol/mg protein in control cells) as well as a transient induction in the level of glucocorticoid receptor (GR) mRNA (4- to 8-fold stimulation after 2-3 h, followed by a decline towards the control value after 6 h). In the presence of the transcription inhibitor actinomycin D (AMD) (5.0 micrograms/ml) the TPA-dependent induction of GR mRNA was completely abolished, and GR mRNA showed a gradual decline with a half-life of 2-3 h. In contrast, treatment with TPA and the protein synthesis inhibitor cycloheximide (50 microM) resulted in a superinduction of GR mRNA (> 50-fold after 6 h). Inhibition of a half-life of 2-3 h, which is identical to that observed in non-treated cells. We conclude that the increase in GR mRNA in the presence of TPA is dependent on ongoing transcription, whereas the rate by which GR transcripts are degraded, is not altered by TPA.

Dactinomycin↗
PubMed1994

Cyclic AMP sensitive signalling by the CD28 marker requires concomitant stimulation by the T-cell antigen receptor (TCR/CD3) complex.

We have previously demonstrated that activation of cAMP-dependent protein kinase (cAK) type I (cAKI, RI alpha 2-C beta 2) mediates the inhibitory effects of cAMP on T-cell replication induced through the TCR/CD3 complex. In the present study we have investigated the effect of cAMP on T-cell DNA synthesis, tyrosine phosphorylation of a 100 kDa protein (pp100) and IL2 mRNA expression, induced through stimulation of the TCR/CD3- and/or the CD28 molecules. Our results demonstrate that tyrosine phosphorylation of pp100 stimulated by anti-CD3 is inhibited by cAMP both in the presence and absence of the phorbol ester PMA, and reflects the changes seen in IL2 mRNA expression and T-cell replication. Combined stimulation with anti-CD3 and anti-CD28, which gives a synergistic response in T-cell replication, gave pp100 phosphorylation and IL2 mRNA expression sensitive to cAMP-dependent inhibition. When PMA was added in addition to anti-CD3 and anti-CD28, the inhibitory effect of cAMP on both T-cell replication and pp100 phosphorylation was completely abolished. The fact that pp100 phosphorylation in response to TCR/CD3-, CD28- and PMA stimulation and cAMP mediated inhibition are identical to the effects of the same stimuli on T-cell proliferation, makes this protein an interesting candidate in downstream signalling from these receptors. In addition, our results are compatible with a model where cAMP, through activation of cAKI, eliminates both the PTK and PKC activating capability of the T-cell receptor at a site(s) proximal to PKC activation. Furthermore, the CD28 molecule which activates PTKs, enters the PTK cascade at a point distal to the target(s) for cAKI action. Therefore, during CD28 signalling PKC activation can be achieved either by TCR/CD3 stimulation (inhibited by cAMP), or directly by PMA (not inhibited by cAMP).

CD28 Antigens↗
PubMed1994

Regulation of growth in a neoplastic B cell line by transfected subunits of 3',5'-cyclic adenosine monophosphate-dependent protein kinase.

cAMP inhibits the proliferation of both normal peripheral blood B and T lymphocytes as well as the proliferation of a human neoplastic B precursor cell line (Reh). To positively show that this is mediated via the the catalytic subunit, C alpha, of cAMP-dependent protein kinase, stably transfected Reh cell lines overexpressing C alpha were established. This was achieved by transfection with a construct confering hygromycin resistance together with a zinc-inducible expression of C alpha from the human metallothionine promoter. C alpha transfected clones were shown to confer a 2- to 2.5-fold zinc-dependent increase in C alpha messenger RNA, immunoreactive C, and phosphotransferase activity. The growth rate of clones transfected with C alpha was retarded, and a zinc-dependent inhibition of cell proliferation was demonstrated in the presence of a small trigger dose of forskolin. In contrast, overexpression of the regulatory subunit I alpha had no effect on cAMP-dependent inhibition of cell proliferation. Furthermore, expression of mutant regulatory subunit I alpha AB, which renders cAMP-dependent protein kinase unresponsive to cAMP, clearly protected against that inhibitory effect of cAMP. These data provides evidence that activation of the C subunit (C alpha) of cAMP-dependent protein kinase mediates the inhibitory action of cAMP on cell proliferation in Reh cells.

B-Lymphocytes↗
PubMed1994

Binding of a member of the NF1 family of transcription factors to two distinct cis-acting elements in the promoter and 5'-flanking region of the human cellular retinol binding protein 1 gene.

We studied the interaction of nuclear proteins with the 5'-flanking and promoter region of the human cellular retinol binding protein 1 (hCRBP1) gene and identified seven specific sequences that interacted with nuclear proteins from liver and prostate. Two of these sequences, footprint 1 (Fp1) and footprint 5 (Fp5), were highly homologous, sharing the core sequence GGCCAAC, which has a certain similarity to the consensus sequence for the NF1 binding site. Competition experiments in gel mobility shift assays and DNasel footprinting indicated that a common protein interacted with both elements. Immunological and biochemical data indicated that this protein belongs to the nuclear factor 1 (NF1) family of transcription factors. The ability of the Fp1 and Fp5 sequences to control gene expression was studied by transient transfections of several cell types. In the wild type promoter, both Fp1 and Fp5 acted as repressors of human (h) CRBP1 gene transcription. Once inserted upstream of the basal promoter from the heterologous p12 gene, the function of both Fp1 and Fp5 was reverted to that of transcriptional activators, although Fp5 exerted only moderate transcriptional activation of the chloramphenicol acetyl transferase (CAT) reporter gene. Hence, the position of these NF1 binding sites and the nature of the flanking sequences appear to direct their effect on transcription. Despite close sequence homology, a common core sequence, and a similar ability to bind nuclear proteins in vitro, these results indicate that Fp1 and Fp5 exert similar regulatory functions but to different levels in vivo. In conclusion, these results indicate that a member of the NF1 family plays a significant role in regulating CRBP1 gene expression.

Animals↗