Structural analysis of cGMP-dependent protein kinase using limited proteolysis

cGMP-dependent protein kinase from bovine lung is labile to specific proteolysis. Limited digestion with chymotrypsin produces a 65,000-dalton monomer and a 16,000-dalton dimer from a 150,000-dalton dimeric enzyme. The larger proteolytic fragment represents the COOH-terminal portion of the enzyme and contains the catalytic site along with the cGMP binding site. The smaller fragment representing the NH2-terminal portion of the enzyme contains the autophosphorylation site and the interchain disulfide bond(s). A model defining the functional domains of cGMP-dependent protein kinase is presented and comparisons with cAMP-dependent protein kinase regulatory subunit are discussed.     

Domain organization of chicken gizzard myosin light chain kinase deduced from a cloned cDNA

Myosin light chain kinases (MLCK) are the most studied of the calmodulin-activated enzymes; however, minimal sequence information is available for the smooth muscle form of the enzyme. The production of an antibody against the enzyme and the use of expression vectors for constructing cDNA libraries have facilitated the isolation of a cDNA for this kinase. The derived amino sequence was found to contain a region of high homology (54%) to the rabbit skeletal muscle enzyme and also very significant homology (35%) to the catalytic subunit of phosphorylase b kinase and cGMP-dependent protein kinase. All of these homologies were found in the known catalytic domains of these enzyme, thus enabling us to predict the location of the catalytic domain for the chicken gizzard myosin light chain kinase. Within the catalytic domain a consensus sequence for an ATP-binding site was located. Subcloning and expression of different regions of the cDNA defined a 192 base pair fragment coding for the calmodulin-binding domain of MLCK. Both of the cAMP-dependent protein kinase phosphorylation sites were identified by sequence homology. A linear model for MLCK is presented placing the various domains in relative position. Northern blot analysis and S1 protection and mapping experiments have revealed that the mRNA for MLCK is 5.5 kilobases in length, but there also exists a second mRNA of 2.7 kilobases that shares a high degree of homology with about 520 base pairs at the 3' end of the cDNA for MLCK.     

Molecular cloning of a cDNA for the catalytic subunit of rabbit muscle phosphorylase phosphatase

A cDNA coding for the catalytic subunit of phosphorylase phosphatase (phosphatase C-I/phosphatase-1c) was cloned from a rabbit muscle cDNA library by screening with oligonucleotide probes. Ten clones were analyzed. The full cDNA sequence of 1395 base pairs contained an open reading frame of 990 base pairs flanked by 3' and 5' noncoding regions of 84 and 321 base pairs, respectively. The DNA sequence (and deduced amino acid sequence) of this cDNA is distinctly different from that of a clone of 1492 base pairs previously reported. Our cDNA is essentially identical to the 1492-base pair clone from residue 182 in the 3' direction, but it is completely different in the 5' direction. Consequently, the amino acid sequence deduced from our cDNA differs by 14 amino acids in the amino terminal from that previously reported and extends for an additional 19 amino acids. Probes to the divergent and common region of our cDNA clone hybridized to an mRNA of the same size by Northern blotting. Thus the cDNA we have isolated appears to code for an isoform of the catalytic subunit of phosphorylase phosphatase.     

Structure of Escherichia coli dnaC. Identification of a cysteine residue possibly involved in association with dnaB protein

The nucleotide sequence of the Escherichia coli dnaC gene and the primary structure of the dnaC protein were determined. The NH2-terminal amino acid sequence of the dnaC protein matched that predicted from the nucleotide sequence of the 735-base pair coding region. The dnaC gene lacks characteristic promoter structures; neither the "Pribnow box" nor the "-35 sequence" was detected within 222 base pairs upstream from the initiator ATG codon. There is, however, a typical Shine-Dalgarno sequence 7-10 base pairs before the ATG codon. An upstream open reading frame, separated by just 2 base pairs from the coding region of dnaC, encodes the COOH-terminal half of the dnaT product (protein i; Masai, H., Bond, M. W., and Arai, K. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1256-1260). The dnaC protein contains 245 amino acids with a calculated molecular weight of 27,894 consistent with the observed value (29,000). Similar to dnaG and dnaT, dnaC uses several minor codons; the significance of these minor codons to the low level expression of the protein product in E. coli cells remains to be determined. The in vitro site-directed mutagenesis method was employed to determine the functional region involved in interaction with dnaB protein. The first cysteine residue located in the NH2-terminal region of the dnaC protein (Cys69) was shown to be important for this activity. Overall sequence homology between dnaC protein and lambda P protein, functionally analogous to the dnaC protein in the lambda phage DNA replication, is not extensive. There are, however, several short stretches of homologous regions including the NH2-terminal eight amino acids and the Cys78 region of dnaC protein.     

Nucleotide sequence of the luxB gene of Vibrio harveyi and the complete amino acid sequence of the beta subunit of bacterial luciferase

The nucleotide sequence of the 1.30-kilobase EcoRI/BglII fragment from Vibrio harveyi carrying the majority of the luciferase beta subunit coding region (luxB gene) has been determined. The EcoRI/BglII fragment was derived from a 4.0-kilobase HindIII fragment carrying both luxA and luxB which was detected in a genomic clone bank based on the expression of bioluminescence from colonies of Escherichia coli carrying V. harveyi HindIII fragments in plasmid pBR322 (Baldwin, T. O., Berends, T., Bunch, T. A., Holzman, T. F., Rausch, S. K., Shamansky, L., Treat, M. L., and Ziegler, M. M. (1984) Biochemistry 23, 3663-3667). The entire alpha subunit coding sequence (luxA gene) and the amino-terminal 13 codons of the beta subunit sequence (luxB gene) were contained on a 1.85-kilobase EcoRI fragment, the sequence of which has been reported (Cohn, D. H., Mileham, A. J., Simon, M. I., Nealson, K. H., Rausch, S. K., Bonam, D., and Baldwin, T. O. (1985) J. Biol. Chem. 260, 6139-6146). The beta subunit coding sequence was found to terminate 972 bases past the start of the luxB coding sequence. The beta subunit had a calculated molecular weight of 36,349 and comprised a total of 324 amino acid residues; the alpha beta dimer had a molecular weight (alpha + beta) of 76,457. There were 27 base pairs separating the stop codon of the beta subunit structural gene and a 340-base open reading frame extending to (and beyond) the distal BglII site. Approximately two-thirds of the beta subunit was sequenced by protein chemical techniques. The amino acid sequence predicted from the DNA sequence, with few exceptions, confirmed the chemically determined sequence, and the measured amino acid composition was in excellent agreement with the composition implied from the DNA sequence.     

HindII and HindIII restriction maps of the attphi80-tonB-trp region of the Escherichia coli genome, and location of the tonB gene.

The HindII and HindIII restriction maps of the attphi80-tonB-trp region of the Escherichia coli chromosome are presented. Analysis of phage DNAs carrying tonB mutations has allowed identification of a 1,730-base pair HindII fragment containing at least part of the tonB gene. This fragment is 4,020 base pairs from the end of trpA, with the total distance from attphi80 to trpA being 6,550 +/- 800 base pairs. Properties of hybrid plasmids containing insertions of various tonB+ restriction fragments suggest that tonB lies completely within the 1,730-base pair fragment. In addition, apparent fusions of beta-galactoside to proteins within the tonB region suggest that the entire region codes for more than one polypeptide.     

The deduced protein sequence of the human carboxypeptidase N high molecular weight subunit reveals the presence of leucine-rich tandem repeats

Human plasma carboxypeptidase N is a 280-kDa tetramer with two high molecular mass (83-kDa) glycosylated subunits which protect the two 50-kDa catalytic subunits and keep them in the circulation. An initial clone for the 83-kDa subunit was obtained by screening two lambda gt11 human liver cDNA expression libraries with antiserum specific for carboxypeptidase N or the 83-kDa subunit. The libraries were rescreened with the labeled cloned cDNA, and the largest clone obtained (2536-base pair insert) was completely sequenced. The deduced protein sequence matched the sequence of several tryptic peptides from the 83-kDa subunit but did not contain the NH2-terminal sequence. The remaining portion of the protein coding sequence was synthesized by the polymerase chain reaction, cloned, and sequenced. The composite cDNA sequence is 2870 base pairs long with an open reading frame of 1608 base pair coding for a protein of 536 amino acids (Mr = 58,762). The protein sequence contains seven potential N-linked glycosylation sites and a threonine/serine-rich region which is a potential site for attachment of O-linked carbohydrate. The most striking feature is a region (residues 68-355) containing 12 leucine-rich tandem repeats of 24 residues with the following consensus sequence: P-X-X-alpha-F-X-X-L-X-X-L-X-X-L-X-L-X-X-N-X-L-X-X-L (X = any amino acid and alpha = aliphatic amino acids, I, L, or V). This repeating pattern is found in the leucine-rich alpha 2-glycoprotein and in other proteins where it might mediate interactions with macromolecules. This region also contains five sequences with heptad repeating leucine residues comprising a leucine zipper motif. The leucine-rich domain likely constitutes an important structural or functional element in the interactions of the 83- and 50-kDa subunits to form the active tetramer of carboxypeptidase N.     

Complete murine cDNA sequence, genomic structure, and tissue expression of the high mobility group protein HMG-I(Y)

A cDNA coding for the non-histone chromosomal protein HMG-I, or its isoform HMG-Y, was isolated from a murine Friend cell library using synthetic oligonucleotide hybridization probes. Sequence analysis showed that the 1670-base pair full length cDNA insert consists of a 201-base pair, G/C-rich (74%), 5'-untranslated region, a 288-base pair amino acid coding sequence, and an unusually long 1182-base pair 3'-untranslated region. The deduced 96-residue amino acid coding sequence of the murine HMG-I(Y) cDNA is very similar to the reported amino acid sequence of human HMG-I, except that it lacks 11 internal amino acids reported in the human protein. Based on Southern blot hybridization analysis of genomic DNA, there appear to be fewer than five copies of HMG-I(Y) genes in the haploid murine genome. These murine HMG-I(Y) genes contain a large (at least 890 base pairs) exon that includes most, or all, of the 3'-untranslated region; whereas the much shorter 5'-untranslated region and amino acid coding sequences are interrupted by at least one intron. A single size class (approximately 1700 nucleotides in murine cells and 2000 nucleotides in human cells) of HMG-I(Y) mRNAs was detected at high levels in total RNA extracts from rapidly dividing, transformed cells, but to a lesser extent, or not at all, in extracts from slowly or non-dividing cells.     

Phosphorylation of mammalian myosin light chain kinases by the catalytic subunit of cyclic AMP-dependent protein kinase and by cyclic GMP-dependent protein kinase

The phosphorylation of the calmodulin-dependent enzyme myosin light chain kinase, purified from bovine tracheal smooth muscle and human blood platelets, by the catalytic subunit of cAMP-dependent protein kinase and by cGMP-dependent protein kinase was investigated. When myosin light chain kinase which has calmodulin bound is phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, 1 mol of phosphate is incorporated per mol of tracheal myosin light chain kinase or platelet myosin light chain kinase, with no effect on the catalytic activity. Phosphorylation when calmodulin is not bound results in the incorporation of 2 mol of phosphate and significantly decreases the activity. The decrease in myosin light chain kinase activity is due to a 5 to 7-fold increase in the amount of calmodulin required for half-maximal activation of both tracheal and platelet myosin light chain kinase. In contrast to the results with the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase cannot phosphorylate tracheal myosin light chain kinase in the presence of bound calmodulin. When calmodulin is not bound to tracheal myosin light chain kinase, cGMP-dependent protein kinase phosphorylates only one site, and this phosphorylation has no effect on myosin light chain kinase activity. On the other hand, cGMP-dependent protein kinase incorporates phosphate into two sites in platelet myosin light chain kinase when calmodulin is not bound. The sites phosphorylated by the two cyclic nucleotide-dependent protein kinases were compared by two-dimensional peptide mapping following extensive tryptic digestion of the phosphorylated myosin light chain kinases. With respect to the tracheal myosin light chain kinase, the single site phosphorylated by cGMP-dependent protein kinase when calmodulin is not bound appears to be the same site phosphorylated in the tracheal enzyme by the catalytic subunit of cAMP-dependent protein kinase when calmodulin is bound. With respect to the platelet myosin light chain kinase, the additional site that was phosphorylated by cGMP-dependent protein kinase when calmodulin was not bound was different from that phosphorylated by the catalytic subunit of cAMP-dependent protein kinase.     

Analysis of the amino acid sequence of the Pseudomonas aeruginosa galactophilic PA-I lectin.

Based on the NH2-terminal 30-amino acid sequence of Pseudomonas aeruginosa galactophilic PA-I lectin, two degenerate primer oligonucleotides were synthesized and used in polymerase chain reaction with the bacterial chromosomal DNA as a template. A predominant DNA fragment of the appropriate size was radiolabeled and used as a probe for screening a P. aeruginosa genomic lambda gt11 library. One positive clone carrying an insert of about 630 base pairs encompassing the entire PA-I lectin gene was isolated and found to contain a 369-base pair open reading frame between an initiation codon (19 base pairs downstream from the insertion site, subsequent to a Shine-Dalgarno sequence) and two consecutive stop codons, followed by an oligo (seven) A sequence, in a partial dyad symmetry. The deduced amino acid sequence shows excellent agreement with the quantitative amino acid analysis and a perfect match with the NH2-terminal amino acid sequence of the purified lectin. It reveals that the PA-I lectin subunit contains 121 amino acids (M(r) 12,754; pI 4.94) with a predominant central hydrophilic core between two hydrophobic domains. Secondary structure algorithms predict that it is rich in beta sheets and contains several highly antigenic epitopes, but no signal peptide. In the carboxyl region a potential glycosylation site (Asn-Asn-Ser) was identified. Comparative analyses of this lectin sequence with those of lectins from other sources, reported in the protein and gene data banks, did not reveal any extensive homology.     

Guanosine cyclic 3',5'-phosphate dependent protein kinase, a chimeric protein homologous with two separate protein families

The amino acid sequence of bovine lung cGMP-dependent protein kinase has been determined by degradation and alignment of two primary overlapping sets of peptides generated by cleavage at methionyl or arginyl residues. The protein contains 670 residues in a single N alpha-acetylated chain corresponding to a molecular weight of 76 331. The function of the molecule is considered in six segments of sequence which may correspond to four folding domains. From the amino terminus, the first segment is related to the dimerizing property of the protein. The second and third segments appear to have evolved from an ancestral tandem internal gene duplication, generating twin cGMP-binding domains which are homologous to twin domains in the regulatory subunits of cAMP-dependent protein kinase and to the cAMP-binding domain of the catabolite gene activator of Escherichia coli. The fourth and fifth segments may comprise one domain which is homologous to the catalytic subunits of cAMP-dependent protein kinase, of calcium-dependent phosphorylase b kinase, and of certain oncogenic viral protein tyrosine kinases. The regulatory, amino-terminal half of cGMP-dependent protein kinase appears to be related to a family of smaller proteins that bind cAMP for diverse purposes, whereas the catalytic, carboxyl-terminal half is related to a family of protein kinases of varying specificity and varying sensitivity to regulators. These data suggest that ancestral gene splicing events may have been involved in the fusion of two families of proteins to generate the allosteric character of this chimeric enzyme.     

Molecular characterization of the interferon-induced 15-kDa protein. Molecular cloning and nucleotide and amino acid sequence

We have isolated a cDNA clone for an interferon-induced 15-kDa protein. The cDNA clone was prepared from mRNA isolated from interferon-beta-treated human Daudi cells. The clone of 635 base pairs contains an open reading frame coding for a protein of 145 amino acids, and suggests for the mRNA a 75-base pair 5' untranslated and a 125-base pair 3' untranslated region. Approximately 85% of the amino acid sequence of the 15-kDa protein has been independently obtained from 2 nmol of material using microsequencing technology on the N terminus of the intact protein and on tryptic and chymotryptic peptides. The amino acid sequence of the isolated protein is identical to the amino acid sequence deduced from the cDNA. Northern blot analysis confirmed that the mRNA for the 15-kDa protein is undetectable in untreated cells, but is greatly induced following interferon treatment.     

Amino acid sequence around a "hinge" region and its "autophosphorylation" site in bovine Lung cGMP-dependent protein kinase

An exposed "hinge" region of cGMP-dependent protein kinase is known to be susceptible to both limited proteolysis and autophosphorylation. A 91-residue fragment has been isolated from this region and its amino acid sequence has been compared with the analogous regions of the cAMP-dependent protein kinases. Although a resemblance among these sequences is not striking, the phosphorylation sites are in corresponding regions toward the NH2 termini, and there are indications of homology in the vicinity of their autophosphorylation sites. As in the cAMP-dependent protein kinase, the site of autophosphorylation and the site of susceptibility to limited proteolysis are very near each other in the primary structure. The actual site of autophosphorylation (the underlined threonine residue in Pro-Arg-Thr-Thr-Arg) is quite different from those in the regulatory subunit of Type II cAMP-dependent kinase or the site in Type I regulatory subunit that can be phosphorylated by the cGMP-dependent protein kinase.     

Cloning and expression of an intron-less gene for AKAP 75, an anchor protein for the regulatory subunit of cAMP-dependent protein kinase II beta.

The A-Kinase Anchor Protein AKAP 75 (formerly designated bovine brain P75) is a particulate brain protein that avidly binds the regulatory subunit (RII beta) of cAMP-dependent protein kinase II beta (Bregman, D. B., Hirsch, A.H. and Rubin, C.S. (1991) J. Biol. Chem. 266, 7207-7213). The formation of stable AKAP 75.RII beta complexes provides a potential mechanism for targeting physiological signals carried by cAMP to specific effector sites within neurons and other brain cells. We have now cloned and characterized the AKAP 75 gene. Its coding sequence is novel and unexpectedly short (1284 base pairs) and contains no introns. When the AKAP 75 gene was transfected into HEK 293 cells, a new RII beta-binding protein with an apparent Mr of 75,000 accumulated. A high proportion (approximately 65%) of the AKAP 75 gene product was excluded from the cytoplasm and was recovered in the 40,000 x g pellet derived from disrupted transfected cells. In contrast, cells transfected with a construct encoding 249 amino acids from the central and C-terminal regions of AKAP 75 produced an RII beta-binding protein (apparent Mr = 45,000) that was exclusively cytosolic. AKAP 75 is a novel protein composed of only 428 amino acid residues (Mr = 47,878). A highly acidic C-terminal region mediates the binding of RII beta (and cAMP-dependent protein kinase II beta), whereas a positively charged N-terminal segment contains structural features that are essential for the association of AKAP 75 with the cytoskeleton and/or intracellular membranes.     

Isolation, nucleotide sequence, and disruption of the Saccharomyces cerevisiae gene encoding mitochondrial NADP(H)-specific isocitrate dehydrogenase

Mitochondrial NADP(H)-specific isocitrate dehydrogenase (IDP1) was purified from yeast cells grown with acetate as a carbon source. IDP1 was shown to be a dimer with a subunit molecular weight of approximately 45,000. Immunochemical levels of IDP1 were found to vary in inverse proportion with those of mitochondrial NAD(H)-specific isocitrate dehydrogenase in cells grown with glucose or with acetate as a carbon source. A 20-residue amino-terminal sequence was obtained for IDP1, and degenerate oligonucleotides were used to synthesize a 50-base pair polymerase chain reaction product corresponding to the coding region for a portion of the amino terminus. The 50-base pair DNA fragment was used as a hybridization probe to identify plasmids containing the IDP1 gene in a yeast genomic DNA library. The complete nucleotide sequence of the IDP1 coding region was determined and translated into a 412-residue amino acid sequence for the mature protein which is preceded by a putative 16-residue mitochondrial targeting presequence. A haploid yeast strain containing a chromosomal disruption of the IDP1 locus was constructed and found to be capable of growth with glucose but not with other carbon sources, suggesting that IDP1 provides a critical function and may be the primary source of NADPH in yeast mitochondria.     

A yeast gene coding for a putative protein kinase homologous to cdc25 suppressing protein kinase

A yeast gene termed YKR coding for a putative protein kinase was isolated by using the cloned cDNA for rabbit protein kinase C as a hybridization probe. The encoded protein (YKR), composed of 380 amino acid residues, shows extensive sequence homology to serine/threonine-specific protein kinases from various species in the approx. 320 C-terminal amino acid residues, strongly suggesting that YKR is endowed with a protein kinase activity. The observed homologies to the cdc25 suppressing protein kinase from yeast, the catalytic subunit of mammalian cAMP-dependent protein kinase, and mammalian protein kinase C were 76, 48 and 37%, respectively. Gene replacement experiments showed that YKR itself is not essential for cell proliferation.