Biomedical subjects

K Taskén

Publications and source records attributed to K Taskén.

80 records · Page 5Linked to original sources

PubMed1991

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

Complementary DNA clones for the regulatory subunit RI beta of cAMP-dependent protein kinases were isolated from a human testis cDNA library using a mouse RI beta 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 RI beta 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 RI beta mRNA were found to be expressed in most tissues, and not restricted to brain and testis as reported by others.

Amino Acid Sequence↗

PubMed1991

Different mechanisms are involved in cAMP-mediated induction of mRNAs for subunits of cAMP-dependent protein kinases.

The present study addresses possible mechanisms through which cAMP mediates its effects on mRNA levels for the subunits of protein kinase A (PKA) and the cellular protooncogene, c-fos. Messenger RNAs for the PKA subunits (RI alpha, RII alpha, RII beta, and C alpha) were regulated by cAMP with similar kinetics in Sertoli cells. However, effects of cAMP on the PKA mRNAs were slow compared to a well characterized cAMP responsive gene, c-fos. The magnitude of stimulation was dramatically different between the various PKA subunits, in that RII beta mRNA increased more than 50-fold while the mRNAs for the other subunits were induced only two to four times. Separation of nuclear and cytoplasmic RNA demonstrated that mRNAs for PKA subunits were stimulated to the same extent in these two cellular compartments. The more rapid induction of c-fos mRNA by cAMP, compared to the mRNA for RII beta, was also seen at the level of transcription. Maximal transcription rate for c-fos, RI alpha, and C alpha were observed after 30 min, whereas that for RII beta was increasing during the 2-h period examined. Transcriptional activation of the RI alpha gene also appeared faster than that for RII beta. When Sertoli cells were incubated with 8-(4-chlorophenylthio) cAMP and cycloheximide, a potent inhibitor of protein synthesis, we observed a super-induction of the mRNAs for c-fos (10-fold) and RI alpha (2-fold).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Protein kinase C activation selectively increases mRNA levels for one of the regulatory subunits (RI alpha) of cAMP-dependent protein kinases in HT-29 cells.

We have examined the effect of the protein kinase C activator, TPA, on mRNA levels for subunits of cAMP-dependent protein kinases in the human colonic cancer cell line HT-29, subline m2. Messenger RNA for the regulatory subunit, RI alpha, of cAMP-dependent protein kinases was shown to be present and regulated by TPA. Other mRNAs for subunits of cAMP-dependent protein kinases (RI beta, RII alpha, RII beta, C alpha, C beta) were also present in these cells, but revealed no or only minor changes upon TPA stimulation. When HT-29 cells were cultured in the presence of 10 nM TPA for various time periods, a biphasic response was observed in RI alpha mRNA levels with a maximal increase (approximately 4 fold) after 24 hours. TPA stimulated RI alpha mRNA increased in a concentration-dependent manner and maximal response (4-8 fold) was seen at 3-10 nM. The TPA-induced increase in RI alpha mRNA was not obtained when cells were incubated with TPA together with the protein kinase C inhibitors, staurosporine or H7. The cAMP-analog 8-CPTcAMP alone induced RI alpha mRNA levels 50% more than TPA. Combined treatment with TPA (10 nM) and 8-CPTcAMP (0.1 mM) gave an increase in RI alpha mRNA similar to TPA. These results demonstrate an interaction between the protein kinase C pathway and mRNA levels for the RI alpha subunit of cAMP-dependent protein kinases in HT-29 cells.

Blotting, Northern↗

PubMed1990

Alloreactive lymphokine-activated killer cells from athymic nude rats do not express CD3-associated alpha/beta or gamma/delta T cell receptors.

Lymphokine activated killer (LAK) cells from athymic nude rats, previously shown to selectively kill MHC-incompatible small lymphocytes in vitro, were examined for rearrangement and expression of genes for the T cell antigen receptors. Southern blots showed no rearrangement of the TCR beta-chain genes, and Northern blots showed transcription only of truncated 1.0 kb beta-chain messages, but not of full-length, 1.3 kb beta-chain mRNA. Transcription of the alpha-chain of the TCR could not be detected, and surface staining with the mAb R73 showed no expression of the rat TCR alpha/beta heterodimer. Transcripts hybridizing with a rat TCR C gamma probe were detected on Northern blots, but probes for all presently characterized mouse V gamma genes failed to hybridize to the same filters, indicating that the C gamma-containing transcripts probably were from non-rearrangement genes. CD3 delta- and epsilon-chain transcripts could not be detected by Northern blot analysis. Less than 2% of the cells stained with the anti-rat CD3 monoclonal antibody 1F4, and incubation with 1F4 had no effect on the alloreactivity of nude rat LAK cells. We have previously shown that immunoglobulin is not involved in the killing of the allogeneic lymphocytes. The most likely interpretation of these results is therefore that nude rat LAK cells express a novel receptor structure involved in allorecognition.

Animals↗

PubMed1988

Molecular cloning, complementary deoxyribonucleic acid structure and predicted full-length amino acid sequence of the hormone-inducible regulatory subunit of 3'-5'-cyclic adenosine monophosphate-dependent protein kinase from human testis.

In this study, we report the isolation and characterization of a full-length cDNA clone for the hormone-inducible regulatory subunit RII beta (formerly called RII51) of type II cAMP-dependent protein kinase from a human testis cDNA library. The cloned cDNA demonstrated tissue-specific expression of RII beta mRNA in human tissues, with the highest mRNA levels in testis and ovary. The isolated human cDNA clone was 3.3 kilobases (kb) in length and contained 166 base pairs (bp) of G/C-rich 5'-noncoding sequence, an open reading frame of 1254 bp and an A/T-rich 3'-nontranslated region containing 1836 bp followed by an 89 nucleotide long poly(A)-tail. The predicted protein contains 418 amino acids including the start methionine, and the estimated mol wt of human RII beta is 53,856. The nucleotide sequence within the open reading frame and the predicted amino acid sequence of human RII beta are highly conserved compared with partial rat RII beta sequences, displaying 91% and 97% similarity, respectively. Codon preference analysis of the cloned cDNA sequence indicated that the two cAMP-binding domains and the hinge region are highly conserved through evolution, whereas the dimerization domain displayed a codon preference pattern indicative of appearance at a later stage of evolution. The isolated human cDNA detected an FSH- and cAMP-inducible mRNA of 3.2 kb in rat Sertoli cells, thus confirming that the cloned cDNA represents the hormone-inducible regulatory subunit of cAMP-dependent protein kinase. This is the first report documenting the isolation of a full-length cDNA clone for the RII beta of cAMP-dependent protein kinase.

Amino Acid Sequence↗

PubMed1988

Molecular cloning and cell-specific expression of newly discovered subunits of cAMP-dependent protein kinases. Implications for different cellular responses to cAMP.

In recent years a multiplicity in isoforms of cAMP-dependent protein kinases has been revealed. Gene products for four different regulatory subunits (RI alpha, RI beta, RII alpha, RII beta) and two different catalytic subunits (C alpha, C beta) have been identified. We hereby present the molecular cloning of rat cDNAs for RII beta, as well as full-length human cDNAs for RII beta and RI alpha. The amino acid sequences deduced from the cDNAs of the regulatory subunits, revealed dissimilarities which were primarily confined to the N-terminal part of the protein. Based on the vital role in testicular function played by gonadotropin-induced activation of cAMP-dependent protein kinases, mRNA levels for the various subunits of cAMP-dependent protein kinase have been studied in rat testis. A clear pattern of cellular localization of mRNAs for the various subunits of cAMP-dependent protein kinase has been demonstrated. Furthermore, stimulation of Sertoli cells by FSH and cAMP elicited a differential response in mRNA levels for various subunits. A dramatic increase (30-40 fold) in the mRNA for RII beta (3.2 kb) was seen with cAMP stimulation, whereas such treatment had minor effects on mRNAs for RI alpha, RII alpha and C alpha. A distinct pattern of expression for various subunits of cAMP-dependent protein kinase was observed during germ cell differentiation. RI alpha and RI beta were expressed at high levels at early stages of spermatogenesis, whereas unique mRNAs for RII alpha and RII beta appeared in post-meiotic germ cells. Altogether, the present results demonstrate specific expression of mRNAs for different subunits of cAMP-dependent protein kinase in different cell types, during hormonal stimulation and during cellular differentiation. This indicates that the individual subunits may confer specific functional properties to the cAMP-dependent protein kinase holoenzyme and to the cAMP signal pathway of the cell.

Animals↗

PubMed1987

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.

Amino Acid Sequence↗

PubMedDate not supplied

Cyclic-AMP-dependent protein kinase (PKA) in testicular cells. Cell specific expression, differential regulation and targeting of subunits of PKA.

LH and FSH regulate via cyclic adenosine 3'5' cyclic monophosphate (cAMP) and cAMP-dependent protein kinase (PKA), steroid biosynthesis is Leydig and Sertoli cells, respectively. Cyclic AMP also regulates a number of different cellular processes such as cell growth and differentiation, ion channel conductivity, synaptic release of neurotransmitters, and gene transcription. The principle intracellular target for cAMP in mammalian cells is the PKA. The fact that this broad specificity protein kinase mediates a number of discrete physiological responses following cAMP engagement, has raised the question of how specificity is maintained in the cAMP/PKA system. Here we describe features of this signaling pathway that may contribute to explain how differential effects of cAMP may be contributed to features of the PKA signaling pathway.

Animals↗