1994 Characterization of in vitro translated human regulatory and catalytic subunits of cAMP dependent protein kinases. (original) (raw)
Related papers
European Journal of Biochemistry, 1994
ull-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 (RIα, RIβ, RIIα and RIIβ) and C (Cα, Cβ and Cγ) 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 (RIα), 54–55 (RIβ), 51 (RIIα) and 53 kDa (RIIβ) 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 RIIα and RIIβ by the C subunit of PKA, revealed a distinct mobility shift of the RIIα subunit on one-dimensional SDS/PAGE (51–54 kDa), but not of RIIβ (53 kDa). Further characterization of the R subunits by two-dimensional SDS/PAGE revealed that RIα was more acidic than RIβ, with pIs of 6.1–6.0 and 6.4–6.2, respectively. Furthermore, the RIIα protein was more basic than RIIβ, 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.
Structure-Function Studies of the cAMP-Dependent Protein Kinase In Vitro and in Intact Cells
There are 518 protein kinase genes in the human genome; this constitutes about 1.7% of all human genes. The cAMP-dependent protein kinase (PKA) serves as the prototypic model for the study of kinases because it contains a conserved catalytic core shared with all eukaryotic kinases, it is the simplest kinase, and it is one of the best-characterized serine/threonine kinases. PKA is ubiquitous in mammals and regulates multiple physiological mechanisms such as the cell cycle, apoptosis, cell motility, energy metabolism, and gene transcription through a well-defined intracellular signaling pathway. While PKA clearly has a central physiological role it is still unclear how PKA mediates multiple physiological mechanisms at the cellular level. Four approaches were used to explore this question using two PKA catalytic subunits, Cα and Cγ, which share 83% identity in primary structure but differ in function. The first approach sought to identify differences in primary structure between Cγ and...
FEBS Letters, 1986
We report here the isolation and sequence of a cDNA for the type II regulatory subunit of the CAMP-dependent protein kinase (CAMP-PK) from a Igt-I 1 cDNA library derived from a porcine epithelial cell line (LLC-PK,). The cDNA was detected by immunological screening using an affinity purified polyclonal antibody for bovine Rn. DNA sequence analysis of the 467 bp EcoRI insert confirmed the identity of the clone, because the deduced amino acid sequence corresponded to the published sequence for the bovine Rn protein. Northern analysis of total RNA from the LLC-PK, cells indicated a single mRNA species of about 6.0 kb, probably derived from a single copy gene. cyclic AMP dependence Protein kinase Regulatory subunit Expression cloning (LLC-PK, cell) 2. MATERIALS AND METHODS 2. I. Cell culture The porcine epithelial cell line LLC-PKi [ 151 was
Isolation of cDNA clones coding for the catalytic subunit of mouse cAMP-dependent protein kinase
Proceedings of the National Academy of Sciences, 1986
mRNA coding for the catalytic (C) subunit of cAMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) was partially purified from bovine testis by polysome immunoadsorption and oligo(dT)-chromatography. This enriched mRNA preparation was used to prepare and differentially screen a cDNA library. One of the selected cDNA clones was shown to hybrid-select mRNA coding for a 40-kDa protein that was specifically precipitated with antibodies to the C subunit. This bovine cDNA clone was then used to isolate a series of mouse cDNA clones that are complementary to the entire mouse C subunit mRNA. The mouse clones code for a protein of 351 animino acids that shows 98% homology to the bovine C subunit and hybridize to a single mRNA of 2.4 kilobases in mouse heart and brain. Southern blot analysis of total genomic DNA suggests that there is a single mouse gene coding for the C subunit. mRNA levels for both the C subunit and the type I regulatory subunit in various mouse tissues and cell lines were quantitated and compared by using singlestranded RNA probes prepared with SP6 polymerase.
Journal of Biological Chemistry, 1996
Two isoforms of the catalytic subunit of cAMPdependent protein kinase, C␣ and C1, are known to be widely expressed in mammals. Although much is known about the structure and function of C␣, few studies have addressed the possibility of a distinct role for the C proteins. The present study is a detailed comparison of the biochemical properties of these two isoforms, which were initially expressed in Escherichia coli and purified to homogeneity. C1 demonstrated higher K m values for some peptide substrates than did C␣, but C1 was insensitive to substrate inhibition, a phenomenon that was observed with C␣ at substrate concentrations above 100 M. C␣ and C1 displayed distinct IC 50 values for the ␣ and  isoforms of the protein kinase inhibitor, protein kinase inhibitor (5-24) peptide, and the type II␣ regulatory subunit (RII␣). Of particular interest, purified type II holoenzyme containing C1 exhibited a 5-fold lower K a value for cAMP (13 nM) than did type II holoenzyme containing C␣ (63 nM). This latter result was extended to in vivo conditions by employing a transcriptional activation assay. In these experiments, luciferase reporter activity in COS-1 cells expressing RII␣ 2 C1 2 holoenzyme was half-maximal at 12-fold lower concentrations of 8-(4-chlorophenylthio)-cAMP and 5-fold lower concentrations of forskolin than in COS-1 cells expressing RII␣ 2 C␣ 2 holoenzyme. These results provide evidence that type II holoenzyme formed with C1 is preferentially activated by cAMP in vivo and suggest that activation of the holoenzyme is determined in part by interactions between the regulatory and catalytic subunits that have not been described previously.
Proceedings of the National Academy of Sciences, 1987
A cDNA clone for a type II regulatory (R) subunit of the cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) was isolated from a rat skeletal muscle library using a specific 47-base oligonucleotide probe. The rat cDNA was 1.2 kilobases (kb) in length and contained an open reading frame of 1.113 kb representing 92% of the coding region of the molecule. Nick-translated rat cDNA was then used to isolate a mouse Rlt cDNA clone from a brain library that contained an open reading frame of 1.143 kb. Because both cDNAs lacked complete coding sequences, the remainder of the R" coding region was obtained from a 15-kb mouse genomic clone. The mouse R" coding region contains 1.2 kb corresponding to a 400-amino acid protein of 51.141 kDa. The mouse cDNA hybridizes to two mRNA species, a 2.4-kb form that was only observed in testis and a 6.0-kb form found in a wide range of tissues, including testis.
Recombinant type I regulatory subunit of the cAMP‐dependent protein kinase is biologically active
FEBS Letters, 1987
The cDNA for the porcine type I regulatory subunit (RI) of the cAMP‐dependent protein kinase (cAMP‐PK) was cloned into two different bacterial expression vectors: pKK223 and pUC18. Recombinant RI was produced by bacteria transformed with either construct, and purified by affinity chromatography. Both the native RI from the pKK223 construct and the RI with an amino terminal extension of eight amino acids from the pUC18 construct were found to be completely native with regard to inhibition of the catalytic subunit activity and cAMP binding.
Archives of Biochemistry and Biophysics, 2002
The Cc and Ca isoforms of the cAMP-dependent protein kinase (PKA) share 83% identity including all critical catalytic and substrate-binding residues defined to date. Compared to Ca, Cc has a different substrate specificity and a selective pseudosubstrate specificity, exhibiting inhibition by regulatory subunits, but not by the protein kinase inhibitor. In these studies, Cc-mediated gene transcription regulation was compared with that of Ca in four cell lines using transient transfection/dual luciferase assays. As compared to Cc, Ca more efficiently activated a cAMP-response element (CRE)-regulated fragment of the human a-glycoprotein hormone promoter which was coupled to a firefly luciferase reporter gene (pGHa-fluc). This occurred in Cos7, Y1, and Kin8 adrenal cells by 23-, 6.5-, and 1.4-fold, respectively. In contrast, Cc, but not Ca, activated the Sp1RE-regulated herpes simplex virus thymidine kinase promoter which was coupled to a Renilla luciferase reporter (pTK-rluc). In Sp1-deficient Sf9 cells, pGHa-fluc expression was maintained for both isoforms, but cotransfection with an Sp1 expression plasmid was necessary and sufficient for activation of pTK-rluc expression by Cc. In all cell lines, cotransfection with a PDK1 expression plasmid enhanced the transcriptional activation of both Ca and Cc (1.5-to 3-fold), while a catalytically inactive PDK1 mutant (PDKÁKD) did not. These results suggest that both Ca and Cc can activate CRE-responsive genes; however, Ca does so with better efficiency than Cc. In contrast to Ca, Cc activates transcription of genes containing pTK-like Sp1RE sites. Activation of different C subunit isoforms can provide a means to diversify cAMP-mediated transcription, possibly affecting cell phenotype.
European Journal of Biochemistry, 1994
Received January 19Eebruary 21, 1994) -EJB 94 005913 8-Piperidino-CAMP has been shown to bind with high affinity to site A of the regulatory subunit of CAMP-dependent protein kinase type I (AI) whereas it is partially excluded from the homologous site (AII) of isozyme I1 [Ggreid, D., Ekanger, R., Suva, R. H., Miller, J. P., and Dmkeland, S. 0. (1989), Eul: J. Biochem. 181,[28][29][30][31]. To further increase this selectivity, the (I?,)and (S,)diastereoisomers of 8-piperidino-CAMP[ S] were synthesized and analyzed for their potency to inhibit binding of 13H]cAMP to site A and site B from type I (rabbit skeletal muscle) and type I1 (bovine myocardium) CAMP-dependent protein kinases.