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 regulation of the regulatory subunit of type II cAMP-dependent protein kinase from rat ovarian granulosa cells

One isoform of the regulatory subunit of type II cAMP-dependent protein kinase (R-II51; Mr = 51,000) and its electrophoretic variants (R-II51.5 and R-II52; Mr = 51,500 and 52,000, respectively) are selectively induced by estradiol and follicle-stimulating hormone (cAMP) in rat ovarian granulosa cells. To ascertain the amino acid sequence of R-II51 and to gain insight into the molecular events regulating the intracellular content of ovarian follicular R-II51, we constructed a lambda gt11 cDNA expression library from poly(A)+ RNA of hormone-primed rat granulosa cells. A 1.5-kilobase (kb) cDNA insert, isolated from a plaque-purified R-II antibody positive bacteriophage clone, selectively bound R-II51 mRNA as demonstrated by analysis of the hybrid-selected translation product. Restriction maps and sequence analyses of the 1.5-kb cDNA insert and of the 1.8- and 2.2-kb cDNA inserts from two additional clones showed overlapping sequences which span a region of 3065 nucleotides in size. The 1.5- and 1.8-kb cDNA inserts each contained poly(A) addition signals (1508 and 1761 nucleotides, respectively), terminal poly(A) sequences, and the entire coding region for R-II51 (1204 nucleotides) except for a small number of nucleotides at the 5' end. The 2.2-kb cDNA insert contained 394 nucleotides of the coding region a long 3' untranslated region and two more poly(A) addition signals (3041 and 3059 nucleotides). An amino acid microsequence surrounding the autophosphorylation site of pure rat ovarian R-II51 agreed with the amino acid sequence deduced from the nucleotide sequence of the cDNA. Northern blot analyses demonstrated two major mRNA species (1.8 and 3.2 kb in size) in hormone-primed rat ovaries which were approximately 10- and 50-fold greater than the R-II mRNA content in rat brain and rat heart, respectively. Southern blot analysis of rat liver DNA indicated that a single gene codes for R-II51 mRNA. Structural differences among rat ovarian R-II51, rat heart R-II54, and the known amino acid sequences of bovine R-II and R-I subunits also indicate that the rat ovarian R-II51 subunit is the product of a distinct gene.     

Molecular cloning and cDNA structure of the regulatory subunit of type I cAMP-dependent protein kinase from rat brain

Complementary DNA (cDNA) clones encoding the regulatory subunit of the type I cAMP-dependent protein kinase (R-I) were isolated by screening of rat brain cDNA libraries. A 1.5-kilobase (kb) cDNA insert containing the entire coding region was sequenced and full amino acid sequence has been deduced from the nucleotide sequence. The clone encodes for a protein of 380 amino acids that shows 97% homology to the bovine R-I subunit. Northern blot analysis demonstrated two major mRNA species (2.8 and 4.4 kb in size) in rat brain and liver.     

Molecular cloning of the cDNA for rat spleen thymosin beta 10 and the deduced amino acid sequence

A rat spleen cDNA library was prepared and employed for the molecular cloning of the cDNA for thymosin beta 10, a peptide that previously had been found to accompany the closely related peptide, thymosin beta 4, in several species of mammals (S. Erickson-Viitanen, S. Ruggieri, P. Natalini, and B. L. Horecker (1983) Arch. Biochem. Biophys. 225, 407-413). First-round screening with a synthetic oligodeoxynucleotide probe yielded 55 positive clones, and sequence analysis of 11 of these clones revealed that they all coded for a peptide containing the thymosin beta 10 sequence, except for an additional arginyl residue at position 39. This peptide, designated thymosin beta 10arg, had been identified previously in rabbit tissues and reported as a variant of thymosin beta 10 (S. Ruggieri, S. Erickson-Viitanen, and B.L. Horecker (1983) Arch. Biochem. Biophys. 226, 388-392). Analysis of the 55 positive clones using a specific oligodeoxynucleotide probe constructed to correspond to the mRNA sequence, including the codon for Arg39, confirmed that they all coded for the amino acid sequence including Arg39. Based on these results, the existence of a molecular species lacking Arg39 is considered unlikely, and we conclude that thymosin beta 10 contains 43, rather than 42, amino acid residues, with identity to thymosin beta 4 in 32 of the 43 residues. We propose that the name thymosin beta 10 be used to refer to the peptide containing Arg39 and that the designation thymosin beta 10arg be dropped. In the cDNA sequence the codons for Ala1 and Ser43 of thymosin beta 10 are flanked by initiator and terminator codons, respectively; thus, both the thymosin beta 4 and thymosin beta 10, which coexist in mammalian cells and tissues, are synthesized without the formation of larger polypeptide precursors.     

Molecular cloning and characterization of the promoter region of the mouse regulatory subunit RII beta of type II cAMP-dependent protein kinase

The promoter and exon 1 of the regulatory subunit (RII beta) of type II cAMP-dependent protein kinase were isolated from a mouse genomic library. The 5'-flanking DNA lacked TATA and CAAT sites but contained GC rich regions typically found in constitutively expressed house keeping genes. Fusion gene constructs, containing RII beta 5'-flanking sequences and the bacterial CAT structural gene, were transfected into NB2a neuroblastoma cells and CHO cells. The NB2a cells expressed high levels of CAT activity. CHO cells expressed CAT activity at 5% of the level seen in the NB2a cells. Transfection of deletion constructs into both cell lines was used to define the core promoter and enhancer elements. The core promoter was situated between bp -291/-121. An enhancer element was located between bp -1426/-1018.     

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.     

Complete amino acid sequence of rat L-type pyruvate kinase deduced from the cDNA sequence

cDNA clones, containing the entire coding region of rat L-type pyruvate kinase, were isolated and their nucleotide sequences were determined by the dideoxy-chain-termination method. The predicted coding region, which spans 543 amino acids, established the complete amino acid sequence of the L-type isozyme of pyruvate kinase for the first time. The deduced amino acid sequence of the L type has one phosphorylation site in its amino terminus and shows about 68% and 48% homologies with M1-type pyruvate kinase of chicken and yeast pyruvate kinase respectively. Domain A exhibits higher homology than domains B and C. The residues in the active site of the L-type enzyme of rats, lying between domains B and A2, are rather different from those of the M1-type enzyme of chickens, but other residues constituting the active site are identical with those of the chicken M1 type except for one amino acid substitution.     

Molecular basis for regulatory subunit diversity in cAMP-dependent protein kinase: crystal structure of the type II beta regulatory subunit

BACKGROUND: Cyclic AMP binding domains possess common structural features yet are diversely coupled to different signaling modules. Each cAMP binding domain receives and transmits a cAMP signal; however, the signaling networks differ even within the same family of regulatory proteins as evidenced by the long-standing biochemical and physiological differences between type I and type II regulatory subunits of cAMP-dependent protein kinase. RESULTS: We report the first type II regulatory subunit crystal structure, which we determined to 2.45 A resolution and refined to an R factor of 0.176 with a free R factor of 0.198. This new structure of the type II beta regulatory subunit of cAMP-dependent protein kinase demonstrates that the relative orientations of the two tandem cAMP binding domains are very different in the type II beta as compared to the type I alpha regulatory subunit. Each structural unit for binding cAMP contains the highly conserved phosphate binding cassette that can be considered the "signature" motif of cAMP binding domains. This motif is coupled to nonconserved regions that link the cAMP signal to diverse structural and functional modules. CONCLUSIONS: Both the diversity and similarity of cAMP binding sites are demonstrated by this new type II regulatory subunit structure. The structure represents an intramolecular paradigm for the cooperative triad that links two cAMP binding sites through a domain interface to the catalytic subunit of cAMP-dependent protein kinase. The domain interface surface is created by the binding of only one cAMP molecule and is enabled by amino acid sequence variability within the peptide chain that tethers the two domains together.     

Human testis cDNA for the regulatory subunit RII alpha of cAMP-dependent protein kinase encodes an alternate amino-terminal region

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

The amino acid sequence of the 24-kDa subunit, an iron-sulfur protein, of rat liver mitochondrial NADH dehydrogenase deduced from cDNA sequence

Antiserum directed against bovine heart mitochondrial NADH dehydrogenase has been used to screen a rat liver cDNA expression library in lambda gt11. The insert cDNA of a positive clone was found to represent the 24-kDa subunit of NADH dehydrogenase by epitope selection using nitrocellulose filter containing the expressed proteins. The amino acid sequence deduced from the nucleotide sequence of the cloned cDNA indicated that the 24-kDa subunit is produced as a precursor with an amino-terminal extension, and that its mature form consists of 217 amino acid residues with a molecular weight of 23,933.     

Expression cloning of a cDNA encoding the type II regulatory subunit of the cAMP-dependent protein kinase

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 lambda gt-11 cDNA library derived from a porcine epithelial cell line (LLC-PK1). The cDNA was detected by immunological screening using an affinity purified polyclonal antibody for bovine RII. 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 RII protein. Northern analysis of total RNA from the LLC-PK1 cells indicated a single mRNA species of about 6.0 kb, probably derived from a single copy gene.     

Primary structure of rat pulmonary surfactant protein D. cDNA and deduced amino acid sequence.

Surfactant protein D (SP-D) is a carbohydrate-binding glycoprotein containing a collagen-like domain that is synthesized by alveolar type II epithelial cells. The complete primary structure of rat SP-D has been determined by sequencing of a cloned cDNA. The protein consists of three regions: an NH2-terminal segment of 25 amino acids, a collagen-like domain consisting of 59 Gly-X-Y repeats, and a COOH-terminal carbohydrate recognition domain of 153 amino acids. There are 6 cysteine residues present in rat SP-D: 2 in the NH2-terminal noncollagenous segment and 4 in the COOH-terminal carbohydrate-binding domain. The collagenous domain contains one possible N-glycosylation site. The protein is preceded by a cleaved, NH2-terminal signal peptide. SP-D shares considerable homology with the C-type mammalian lectins. Hybridization analysis demonstrates that rat SP-D is encoded by a 1.3-kilobase mRNA which is abundant in lung and highly enriched in alveolar type II cells. Extensive homology exists between rat SP-D and bovine conglutinin.     

The regulatory subunit of the type II cAMP-dependent protein kinase in rabbit ovaries is the RII beta isoform.

Based on RII autophosphorylation, photoaffinity labeling with 8-N3[32P]cAMP, and Western blot analysis we have identified the RII isoform found in rabbit corpora lutea as RII beta. The RII beta subunit found in rabbit corpora lutea differs from the RII beta found in rat follicles and corpora lutea in that it migrates at Mr 52,500 on SDS-PAGE and shifts to Mr 53,000 when phosphorylated.     

Identification of the MAP2- and P75-binding domain in the regulatory subunit (RII beta) of type II cAMP-dependent protein kinase. Cloning and expression of the cDNA for bovine brain RII beta

cDNA clones coding for the regulatory subunit (RII beta) of type II cAMP-dependent protein kinase were isolated from a bovine brain cDNA expression library in lambda gt11. The cDNA codes for a protein of 418 amino acids which is 98% homologous to the rat and human RII beta proteins. A series of expression vectors coding for truncated RII beta proteins were constructed in pATH plasmids and fusion proteins were expressed in Escherichia coli. Polyclonal and monoclonal antibodies made against purified bovine brain RII were immunoreactive with the fusion proteins on Western blots. The expressed RII beta-fusion proteins were used in overlay assays to identify the region in RII beta which binds to microtubule-associated protein 2 (MAP2) and to the 75,000-dalton calmodulin-binding protein (P75) (Sarkar, D., Erlichman, J., and Rubin, C.S. (1984) J. Biol. Chem. 259, 9844-9846) in bovine brain. Fusion protein containing amino acids 1-50 of the RII beta NH2 terminus (RII beta(1-50)] bound to both MAP2 and P75 immobilized on nitrocellulose filters. A pATH11-directed fusion protein containing the 31 amino acid RII-binding site of the human MAP2 protein (MAP2(31)) (Rubino, H.M., Dammerman, M., Shafit-Zagardo, B., and Erlichman, J. (1989) Neuron 3, 631-638) also bound RII beta-fusion proteins containing RII beta amino acids 1-50. Three fusion proteins, RII beta(1-25), RII beta(25-96), and RII beta(1-265,25-96 deleted) did not bind to MAP2(31) nor P75. The results showed that the binding domain for MAP2 and P75 was located within the NH2-terminal 50 amino acids of RII beta. Preincubation of bovine heart protein kinase II alpha and RII beta(1-50) with MAP2(31) prevented their binding to both P75 and MAP2(31) that were immobilized on nitrocellulose, suggesting that the binding sites for MAP2 and P75 are located near each other or that the same site on RII was binding to both proteins.     

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.     

Complete amino acid sequence of Bombyx egg-specific protein deduced from cDNA clone

Complementary (c)DNA coding for an insect yolk protein, the egg-specific protein of the silkworm Bombyx mori was cloned and the nucleotide sequence determined. The sequence covers the entire coding region of 1,677 base pairs with 5′ and 3′ noncoding regions (21 and 115 base pairs, respectively). The deduced amino acid sequence of the egg-specific protein consists of 559 amino acid residues. The NH₂-terminal 18 amino acid sequence is enriched in hydrophobic amino acids and assumed to be a signal peptide. A sequence, Asn-X-Thr, a potential N-linked glycosylation site, is found at positions 191 to 193. A serine-rich domain is localized in the region from 63 to 90, in which phosphorylation takes place. Cys His motif in 405 to 415 is analogous to a proposed metal binding sequence. Lys¹³²-Asn¹³³ and Arg²²⁸-Asp²²⁹ are probably the sites cleaved by the egg-specific protein protease that appears during embryogenesis. The derived amino acid sequence has no appreciable homology to other sequenced proteins.     

The amino acid sequence of rat liver glucokinase deduced from cloned cDNA

Rat liver glucokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) was purified to homogeneity, cleaved, and subjected to amino acid sequence analysis. Forty-five percent of the protein sequence was obtained, and this information was used to design oligonucleotide probes to screen a rat liver cDNA library. A 1601-base pair cDNA (GK1) contained an open reading frame that encoded the amino acid sequences found in the peptides used to generate the oligonucleotide probes. A second cDNA was subsequently identified (GK.Z2), which is 2346 base pairs long and corresponds to nearly the entire glucokinase mRNA. Blot transfer analysis of hepatic RNA showed that glucokinase mRNA exists as a single species of about 2400 nucleotides. Four hours of insulin treatment of diabetic rats resulted in a 30-fold induction of this mRNA. GK.Z2 has a long open reading frame which, with the known partial peptide sequence, allowed us to deduce the primary structure of glucokinase. The enzyme is composed of 465 amino acids and has a mass of 51,924 daltons. Glucokinase has 53 and 33% amino acid sequence identities with the carboxyl-terminal domains of rat brain hexokinase I and yeast hexokinase, respectively. If conservative amino acid replacements are also considered, glucokinase is similar to these two enzymes at 75 and 63% of positions, respectively. The putative glucose- and ATP-binding domains of glucokinase were identified, and these regions appear to be highly conserved in the hexokinase family of enzymes.     

Complete amino acid sequence of rat liver alcohol dehydrogenase deduced from the cDNA sequence

Alcohol dehydrogenase (ADH) catalyzes the rate-determining reaction in the metabolism of ethanol. We report here the complete nucleotide sequence of a cDNA encoding rat liver ADH, and the deduced amino acid (aa) sequence of the protein. The rat enzyme contains a cluster of aa substitutions and an aa insertion in the region between aa residues 111 and 118, which is near the intron-exon junction reported for the human ADH gene. It also contains an additional cysteine in the highly variable region from aa residues 108-125 which may account for the unusual lability of rat ADH compared with ADH from other species.