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Biomedical subjects

A R Means

Publications and source records attributed to A R Means.

At least 109 records · Page 6Linked to original sources
PubMed1989

Myosin light chain kinase structure function analysis using bacterial expression.

A 40-kDa fragment of chicken smooth muscle myosin light chain kinase was produced and partially purified from a bacterial expression system. This fragment exhibits calmodulin binding and substrate phosphorylation properties similar to those of the isolated chicken gizzard enzyme. A series of 3'-deletion mutants was prepared and used to produce proteins with the same NH2 terminus but with COOH termini varying over 180 amino acids. Results show that truncation of the enzyme at Ser-512 (based on the amino acid numbering system described for the partial cDNA clone by Guerriero, V., Jr., Russo, M. A., Olson, N. J., Putkey, J. A., and Means, A. R. (1986) Biochemistry 25, 8372-8381) does not alter calmodulin binding, calmodulin regulation, or enzymatic properties. Removal of an additional 5 residues from the COOH terminus completely inhibits calmodulin binding and results in an inactive kinase that can be fully activated by limited proteolysis. Site specific mutations within these 5 residues demonstrate that Gly-508 and Arg-509 are independently involved in calmodulin-dependent binding and activation of myosin light chain kinase. Truncation of the enzyme at residues within the protein kinase catalytic domain results in inactive protein that cannot be activated by proteolysis.

Animals↗
PubMed1989

Calmodulin-induced early-onset diabetes in transgenic mice.

Calmodulin is implicated as the primary transducer of the calcium signal in pancreatic beta cells, where it is present at very high concentrations. We have produced three lines of transgenic mice carrying a calmodulin minigene regulated by the rat insulin II promoter. Immunohistochemistry and hybridization analyses indicated a 5-fold increase in the content of calmodulin and its mRNA in beta cells. Transgenic mice developed severe diabetes within hours of birth. The diabetes was progressive and characterized by elevated blood glucose and glucagon that coincided with reduced serum and pancreatic insulin. The disease appears to have both secretory and cell destruction components. beta Cell pathology was evident in the diabetic neonate but not in the fetus, suggesting that the early onset of diabetes exacerbated possible toxic effects of excess calmodulin. These animals provide a new diabetes model to evaluate how abnormal calcium homeostasis alters the carefully programmed secretory activity and development of the beta cell.

Aging↗
PubMed1989

Changes in the structure of calmodulin induced by a peptide based on the calmodulin-binding domain of myosin light chain kinase.

Small-angle X-ray and neutron scattering data were used to study the solution structure of calmodulin complexed with a synthetic peptide corresponding to residues 577-603 of rabbit skeletal muscle myosin light chain kinase. The X-ray data indicate that, in the presence of Ca2+, the calmodulin-peptide complex has a structure that is considerably more compact than uncomplexed calmodulin. The radius of gyration, Rg, for the complex is approximately 20% smaller than that of uncomplexed Ca2+.calmodulin (16 vs 21 A), and the maximum dimension, dmax, for the complex is also about 20% smaller (49 vs 67 A). The peptide-induced conformational rearrangement of calmodulin is [Ca2+] dependent. The length distribution function for the complex is more symmetric than that for uncomplexed Ca2+.calmodulin, indicating that more of the mass is distributed toward the center of mass for the complex, compared with the dumbell-shaped Ca2+.calmodulin. The solvent contrast dependence of Rg for neutron scattering indicates that the peptide is located more toward the center of the complex, while the calmodulin is located more peripherally, and that the centers of mass of the calmodulin and the peptide are not coincident. The scattering data support the hypothesis that the interconnecting helix region observed in the crystal structure for calmodulin is quite flexible in solution, allowing the two lobes of calmodulin to form close contacts on binding the peptide. This flexibility of the central helix may play a critical role in activating target enzymes such as myosin light chain kinase.

Animals↗
PubMed1989

Molecular cloning sequence and distribution of rat calspermin, a high affinity calmodulin-binding protein.

Calspermin is a heat-stable, acidic calmodulin-binding protein predominantly found in mammalian testis. The cDNA representing the rat form of this protein has been cloned from a rat testis lambda gt11 library. Sequence analysis of two overlapping clones revealed a 232-nucleotide 5'-nontranslated region, 510 nucleotides of open reading frame, a 148-nucleotide 3'-untranslated region, and a poly(A) tail. Authenticity of the clones was confirmed by comparison of a portion of the deduced amino acid sequence with the sequence of a tryptic peptide obtained from the rat testis protein. The lambda gt11 fusion protein was recognized by affinity purified antibodies to pig testis calspermin and bound 125I-calmodulin in a Ca2+-dependent manner. Calspermin cDNA encodes a 169-residue protein with a calculated Mr of 18,735. The putative calmodulin-binding domain is very close to the amino terminus of the protein. This region shows 46% identity with the calmodulin-binding region of rat brain Ca2+/calmodulin-dependent protein kinase II and 32% identity with the equivalent region of chicken smooth muscle myosin light chain kinase. The 5'-nontranslated region reveals significant homology with a portion of the catalytic region of the calmodulin-dependent protein kinase family. Calspermin contains a stretch of 17 contiguous glutamic acid residues in the central region of the molecule. Computer analysis predicts calspermin to be 81% alpha-helix and 14% random coil. Analysis of genomic DNA indicates calspermin to be the product of a unique gene. Northern blot analysis of rat testis RNA reveals a 1.1-kilobase mRNA. This RNA is restricted to testis among several rat tissues examined and could not be identified in total RNA isolated from testes of other mammals. Analysis of cells isolated from rat testis reveals calspermin mRNA to be predominantly expressed in postmeiotic cells indicating that it may be specific to haploid cells.

Amino Acid Sequence↗
PubMed1989

Calmodulin is required for cell-cycle progression during G1 and mitosis.

In order to examine the consequences of a transient increase or decrease in intracellular calmodulin (CaM) levels, two bovine-papilloma-virus (BPV)-based expression vectors capable of inducibly synthesizing CaM sense (BPV-MCM) or anti-sense (BPV-CaMAS) RNA have been constructed and used to stably transform mouse C127 cells. Upon addition of Zn2+, cells containing the BPV-MCM vector have transiently increased CaM mRNA and protein levels. Cells carrying the BPV-CaMAS vector transiently produce CaM anti-sense RNA resulting in a significant decrease in intracellular CaM concentration. Increased CaM caused a transient acceleration of proliferation, while the anti-sense RNA induced decrease in CaM caused a transient cell cycle arrest. Flow cytometric analysis showed that progression through G1 and mitosis was affected by changes in CaM levels. These data indicate that CaM levels may limit the rate of cell-cycle progression under normal conditions of growth.

Animals↗
PubMed1989

Calmodulin, cell growth and gene expression.

Calmodulin is thought to regulate a number of intracellular processes, including cell proliferation. Previous studies using drugs that antagonize calmodulin function have indicated that calmodulin is required for progression at specific points in the eukaryotic cell cycle. However, interpretation of these results has previously been difficult due to the lack of specificity of these inhibitors in living cells. Recent studies have used a combination of molecular biological and genetic analysis techniques to approach the study of calmodulin-dependent cell cycle control with greater precision and specificity. These studies have confirmed that calmodulin is an important regulator of the cell cycle, and provide new ways in which to examine the cellular mechanisms involved in calmodulin-dependent cell cycle control.

Animals↗
PubMed1989

Expression of RNAs for calmodulin, actins, and tubulins in rat testis cells.

Messenger RNAs encoding calmodulin, alpha- and beta-tubulins, and actins were analyzed in nucleic acid isolated from purified rat testis cell populations. Cell-specific patterns were discovered that suggest coordinate regulation of members of different gene families. Genes that are candidates for coordinate regulation include those encoding 1.4 kb calmodulin RNA and 1.8 kb alpha-tubulin RNAs; 1.6 kb calmodulin RNA and a 1.8 kb beta-tubulin RNA; and 1.6 kb actin and 2.1 kb alpha-tubulin RNAs. The steady state level of the 2.2 kb actin RNA(s) varies less than any other RNA analyzed. Actin RNAs of approximately 1.6 kb are observed in fractions enriched for A spermatogonia or round spermatids or cytoplasts shed from elongating spermatids. The level of 1.8 kb alpha-tubulin RNA increases in a fraction enriched for leptotene-zygotene spermatocytes and peaks in pachytene spermatocytes. As the level of this alpha-tubulin RNA species decreases in maturing spermatids a new 2.1 kb alpha-tubulin RNA increases. beta-Tubulin RNAs of 2.7 and 1.8 kb are present in somatic and premeiotic cell fractions. beta-Tubulin RNA transcripts of 1.8 kb that increase in pachytene spermatocytes are derived from a different gene than that expressed in premeiotic cells. The major RNA that hybridizes to the chicken calmodulin cDNA is 1.4 kb; it increases during the development of spermatocytes, reaching a maximum level in pachytene spermatocytes before it declines during spermiogenesis. Calmodulin RNAs of 1.6 and 1.0 kb are clearly evident in the round spermatid fraction. These data reveal that mechanisms exist for cell-selective elevation of mRNAs encoding cytoskeletal proteins during rat spermatogenesis.

Actins↗
PubMed1989

Chicken calmodulin promoter activity in proliferating and differentiated cells.

A 1218-base pair (bp) portion of the chicken calmodulin promoter was sequenced and assayed for promoter activity. This portion of the promoter was found sufficient to produce accurate transcriptional initiation. The promoter sequence was GC rich, particularly in the 700 bp region 5' to the cap site. Eight plasmids were prepared containing the first calmodulin exon and 30-1218 bp of the promoter, ligated to the reporter gene chloramphenicol acetyl transferase. In chicken embryonic fibroblasts and proliferating BC3H-1 cells promoter activity increased progressively with increasing promoter length up to 617 bp. Extension of the promoter beyond 617 bp inhibited expression, as did sequences within the first calmodulin exon. In BC3H-1 cells differentiation was found to reduce calmodulin mRNA levels approximately 3-fold. Activity of the calmodulin promoter constructs also decreased by a similar extent with differentiation. Sequences up to 234 bp 5' to the calmodulin cap site were markedly less effective in elevating chloramphenicol acetyl transferase activity in differentiated BC3H-1 cells than in proliferating cells and may account for the lower overall activity of the calmodulin promoter in these cells. Within this region several sequences were identified, including an extensive homology to the rat calmodulin I gene promoter that could be significant in regulation of calmodulin expression.

Animals↗
PubMed1989

Analysis of expression of multiple genes encoding calmodulin during spermatogenesis.

It has recently been determined that the intracellular calcium receptor, calmodulin, is encoded by a multigene family. At least three calmodulin genes that encode the identical protein are expressed in adult testis from a variety of mammalian species. The complementary techniques of RNA blot hybridization and in situ hybridization were used to assess the relative levels of calmodulin RNAs during rat testis cell differentiation. RNA isolated from highly purified populations of somatic or germinal cells and sections of fixed testis were analyzed for hybridization to specific probes corresponding to the three separate rat calmodulin genes. The level of each calmodulin RNA can be described by a unique developmental pattern during spermatogenesis. The steady state levels of transcripts corresponding to calmodulin gene I (CaM I) and III increase in early meiotic cells (leptotene-zygotene spermatocytes) and remain constant throughout meiosis. The level of the CaM II RNA increases between early and mid-pachytene spermatocytes, but is only transiently elevated. The level of this RNA decreases in round spermatids and is almost nondetectable in cytoplasts shed from elongating spermatids. The levels of CaM I RNAs are maintained in early round spermatids, but only RNA derived from the CaM III gene is still evident in late spermatids. The data indicate that an individual germ cell contains RNAs derived from multiple calmodulin genes and suggests that the divergent calmodulin genes may respond to different cellular regulatory signals.

Animals↗
PubMed1989

The presence of parvalbumin in a nonmuscle cell line attenuates progression through mitosis.

Based on studies that have examined the effect of calcium chelators on cells, it has been proposed that this cation plays a role in regulating cell proliferation. In this study a novel approach was used to indirectly examine the role of calcium in cell cycle progression. A cDNA for the Ca2+-binding protein parvalbumin has been expressed in mouse C127 cells, using a bovine papilloma virus-based expression vector. The normal role of parvalbumin is that of a calcium buffer in vertebrate fast twitch muscle, and the C127 cells do not normally express this protein. The presence of parvalbumin had several effects on the growth of C127 cells. The most striking phenotype was an increase in cell cycle duration which analysis showed was the result of an increase the length of G1 and mitosis (predominantly at prophase). Since changes in cell cycle duration typically occur as a result of changes in G1 duration, the observed increase in the length of mitosis is most unusual. The present results indicate that the previously observed increase in the rate of cell proliferation in cells with elevated calmodulin levels is not the result of a general increase in the level of cytoplasmic calcium-binding protein, but is specific to calmodulin. In addition, the results suggest that calcium regulates progression through mitosis by both calmodulin-dependent (metaphase transition) and -independent (prophase) mechanisms.

Animals↗
PubMed1988

Domain II of calmodulin is involved in activation of calcineurin.

A family of mutant proteins related to calmodulin (CaM) has been produced using cDNA constructs in bacterial expression vectors. The new proteins contain amino acid substitutions in Ca2+-binding domains I, II, both I and II, or both II and IV. The calmodulin-like proteins have been characterized with respect to mobility on SDS-polyacrylamide gels, Ca2+-dependent enhancement of tyrosine fluorescence, and abilities to activate the CaM-dependent phosphatase calcineurin. These studies suggest that an intact Ca2+-binding domain II is minimally required for full activation of calcineurin.

Animals↗
PubMed1988

Autoregulation of enzymes by pseudosubstrate prototopes: myosin light chain kinase.

The myosin light chain kinase requires calmodulin for activation. Tryptic cleavage of the enzyme generates an inactive 64-kilodalton (kD) fragment that can be further cleaved to form a constitutively active, calmodulin-independent, 61-kD fragment. Microsequencing and amino acid analysis of purified peptides after proteolysis of the 61- and 64-kD fragments were used to determine the amino-terminal and carboxyl-terminal sequences of the 64-kD fragment. Cleavage within the calmodulin-binding region at Arg505 generates the catalytically inactive 64-kD fragment, which is incapable of binding calmodulin. Further digestion removes a carboxyl-terminal fragment, including the pseudosubstrate sequence Ser484-Lys-Asp-Arg-Met-Lys-Lys-Tyr-Met- Ala-Arg-Arg-Lys-Trp-Gln-Lys-Thr-Gly-His-Ala-Val-Arg505 and results in a calmodulin-independent 61-kD fragment. Both the 61- and 64-kD fragments have the same primary amino-terminal sequences. These results provide direct support for the concept that the pseudosubstrate structure binds the active site and that the role of calmodulin is to modulate this interaction. Pseudosubstrates may be utilized in analogous ways by other allosterically regulated enzymes.

Amino Acid Sequence↗
PubMed1988

Functional significance of the central helix in calmodulin.

The 3-A crystal structure of calmodulin indicates that it has a polarized tertiary arrangement in which calcium binding domains I and II are separated from domains III and IV by a long central helix consisting of residues 65-92. To investigate the functional significance of the central helix, mutated calmodulins were engineered with alterations in this region. Using oligonucleotide-primed site-directed mutagenesis, Thr-79 was converted to Pro-79 to generate CaMPM. CaMPM was further mutated by insertion of Pro-Ser-Thr-Asp between Asp-78 and Pro-79 to yield CaMIM. Calmodulin, CaMPM, and CaMIM were indistinguishable in their ability to activate calcineurin and Ca2+-ATPase. All mutated calmodulins would also maximally activate cGMP-phosphodiesterase and myosin light chain kinase, however, the concentrations of CaMPM and CaMIM necessary for half-maximal activation (Kact) were 2- and 9-fold greater, respectively, than CaM23. Conversion of the 2 Pro residues in CaMIM to amino acids that predict retention of helical secondary structure did not restore normal calmodulin activity. To investigate the nature of the interaction between mutated calmodulins and target enzymes, synthetic peptides modeled after the calmodulin binding region of smooth and skeletal muscle myosin light chain kinase were prepared and used as inhibitors of calmodulin-dependent cGMP-phosphodiesterase. The data suggest that the different kinetics of activation of myosin light chain kinase by CaM23 and CaMIM are not due to differences in the ability of the activators to bind to the calmodulin binding site of this enzyme. These observations are consistent with a model in which the length but not composition of the central helix is more important for the activation of certain enzymes. The data also support the hypothesis that calmodulin contains multiple sites for protein-protein interaction that are differentially recognized by its multiple target proteins.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1988

Calcium/calmodulin-dependent protein kinase II. Characterization of distinct calmodulin binding and inhibitory domains.

Regulatory domains of the multifunctional Ca2+/calmodulin-dependent protein kinase II were investigated utilizing synthetic peptides. These peptides were derived from the sequence between positions 281 and 319 as translated from the cDNA sequence of the rat brain 50-kDa subunit (Lin, C. R., Kapiloff, M. S., Durgerian, S., Tatemoto, K., Russo, A. F., Hanson, P., Schulman, H., and Rosenfeld, M. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5962-5966), which contain the putative calmodulin-binding region as well as potential autophosphorylation sites. Peptide 290 to 309 was found to be a potent calmodulin antagonist with an IC50 of 52 nM for inhibition of Ca2+/calmodulin-dependent protein kinase II. Neither truncation from the amino terminus (peptide 296-309) nor extension in the carboxyl-terminal direction (peptide 294-319) markedly affected calmodulin binding, whereas shortening the peptide from the carboxyl terminus (peptide 290-302) or from both ends (peptide 295-304) resulted in the elimination of this activity. Peptide 281-290 did not bind calmodulin, but was a good substrate for the enzyme, being phosphorylated at Thr-286. Several of the peptides inhibited the kinase in a partially competitive, substrate-directed manner, but were not themselves phosphorylated. These studies identify domains within Ca2+/calmodulin-dependent protein kinase II which may be involved in 1) inhibition of the kinase in the absence of calmodulin, 2) binding of calmodulin, and 3) the resulting activation. Additionally, it is suggested that phosphorylation of residues flanking these domains may be responsible for the known regulatory effects of autophosphorylation on the properties of the kinase.

Amino Acid Sequence↗