Amino acid sequence of the regulatory subunit of bovine type I cAMP-dependent protein kinase (original) (raw)
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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.
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
CAMP-dependent protein kinase: prototype for a family of enzymes
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1988
Protein kinases represent a diverse family of enzymes that play critical roles in regulation. The simplest and best-understood biochemically is the catalytic (C) subunit of cAMP-dependent protein kinase, which can serve as a framework for the entire family. The amino-terminal portion of the C subunit constitutes a nucleotide binding site based on affinity labeling, labeling of lysines, and a conserved triad of glycines. The region beyond this nucleotide fold also contains essential residues. Modification of Asp 184 with a hydrophobic carbodiimide leads to inactivation, and this residue may function as a general base in catalysis. Despite the diversity of the kinase family, all share a homologous catalytic core, and the residues essential for nucleotide binding or catalysis in the C subunit are invariant in every protein kinase. Affinity labeling and intersubunit cross-linking have localized a portion of the peptide binding site, and this region is variable in the kinase family. The ...
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...
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 Molecular Evolution, 2002
The members of the PKA regulatory subunit family (PKA-R family) were analyzed by multiple sequence alignment and clustering based on phylogenetic tree construction. According to the phylogenetic trees generated from multiple sequence alignment of the complete sequences, the PKA-R family was divided into four subfamilies (types I to IV). Members of each subfamily were exclusively from animals (types I and II), fungi (type III), and alveolates (type IV). Application of the same methodology to the cAMP-binding domains, and subsequently to the region delimited by -strands 6 and 7 of the crystal structures of bovine RI␣ and rat RII (the phosphate-binding cassette; PBC), proved that this highly conserved region was enough to classify unequivocally the members of the PKA-R family. A single signature sequence,