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Biomedical subjects

A R Means

Publications and source records attributed to A R Means.

At least 127 records · Page 7Linked to original sources
PubMed1988

Mapping of calmodulin-binding domain of Ca2+/calmodulin-dependent protein kinase II from rat brain.

Recent molecular cloning experiments have identified a 25 amino-acid region as the calmodulin-binding domain of the alpha-subunit of rat brain Ca2+/calmodulin-dependent multifunctional protein kinase II (CaM-K II). Synthetic peptides, derived from the deduced amino-acid sequence encompassing this region, were examined for their ability to bind calmodulin in a calcium dependent manner and to inhibit the Ca2+/calmodulin-dependent autophosphorylation of CaM-K II. Comparison of these structure-function relationships highlighted a region of 5 amino-acids, which was essential for calmodulin interaction and inhibition of kinase activity. This region demonstrated some homology with other calmodulin-binding peptides, and may represent a key site of interaction of the kinase with calmodulin. These analyses provide additional insight into the molecular mechanism underlying the Ca2+ regulation of CaM-K II.

Amino Acid Sequence↗
PubMed1988

Calcium, calmodulin and cell proliferation.

Calcium and calmodulin have been proposed to be regulatory factors in cell cycle progression. Clonal mouse cell lines harboring episomally-carried genes have been prepared to address this question. Some lines produce extra calmodulin, others express antisense RNA to decrease calmodulin, while others produce the Ca2+-buffering protein parvalbumin. The results show that calmodulin acts at two points in the cell cycle--the G1/S boundary and metaphase transition. An additional Ca2+ event that is calmodulin-independent occurs at mitotic prophase. The elevated (or depressed) level of intracellular Ca2+ binding protein does not markedly affect gene expression. In cells containing excess calmodulin, the synthesis mechanisms that normally control the level of calmodulin post-transcriptionally are overridden. Genes normally expressed in G1 whose products are involved in growth control show increases in calmodulin over producing cell lines. Elevated calmodulin decreases tubulin mRNA presumably due to its effect on microtubule stability. The availability of cell lines in which calmodulin can be inducibly increased or decreased should provide tools to elucidate the molecular mechanisms that govern the regulatory roles for this protein in cell cycle progression.

Animals↗
PubMed1988

Identification, characterization, and functional correlation of calmodulin-dependent protein phosphatase in sperm.

Preliminary data demonstrated that the inhibition of reactivated sperm motility by calcium was correlated with inhibited protein phosphorylation. The inhibition of phosphorylation by Ca2+ was found to be catalyzed by the calmodulin-dependent protein phosphatase (calcineurin). Sperm from dog, pig, and sea urchin contain both the Ca2+-binding B subunit of the enzyme (Mr 15,000) and the calmodulin-binding A subunit with an Mr of 63,000. The sperm A subunit is slightly higher in Mr than reported for other tissues. Inhibition of endogenous calmodulin-dependent protein phosphatase activity with a monospecific antibody revealed the presence of 14 phosphoprotein substrates in sperm for this enzyme. The enzyme was localized to both the flagellum and the postacrosomal region of the sperm head. The flagellar phosphatase activity was quantitatively extracted with 0.6 M KCl from isolated flagella from dog, pig, and sea urchin sperm. All salt-extractable phosphatase activity was inhibited with antibodies against the authentic enzyme. Preincubation of sperm models with the purified phosphatase stimulated curvolinear velocity and lateral head amplitude (important components of hyperactivated swimming patterns) and inhibited beat cross frequency suggesting a role for this enzyme in axonemal function. Our results suggest that calmodulin-dependent protein phosphatase plays a major role in the calcium-dependent regulation of flagellar motility.

Animals↗
PubMed1988

Calmodulin regulation of smooth-muscle myosin light-chain kinase.

Calmodulin is the predominant Ca2+ receptor in all nonmuscle and smooth muscle cells. As such, it mediates the activity of more than 20 intracellular enzymes. The structure of calmodulin reveals an eight-turn helix that separates the two pairs of Ca2+ binding sites. This central region is involved in enzyme recognition and/or activation. Now that the sequence of several calmodulin-dependent enzymes is known, it has been revealed that the sequence of each calmodulin binding region is unique but bind calmodulin with equal affinity. The amino-terminal portion of this region in calmodulin-dependent protein kinases serves to inhibit the enzyme from binding substrate in the absence of calmodulin. This pseudosubstrate region is both unique and specific for each enzyme. Therefore calmodulin derepresses rather than activates protein kinases. Studies with smooth-muscle myosin light-chain kinase have identified the pseudosubstrate region. It is proposed that inhibitors directed toward this intramolecular interaction should provide novel drugs for the treatment of a variety of cardiovascular diseases that result in elevated systemic vascular resistance.

Animals↗
PubMed1987

Calmodulin is involved in regulation of cell proliferation.

A chicken calmodulin (CaM) gene has been expressed in mouse C127 cells using a bovine papilloma virus (BPV)-based vector (BPV-CM). The vector-borne genes produce a mature mRNA of the expected size that is present on cytoplasmic polyribosomes. In clonal cell lines transformed by BPV-CM, expression of the CaM gene produced CaM levels 2- to 4-fold above those observed in cells transformed by BPV alone. Increased intracellular CaM caused a reduction of cell cycle length that is solely due to a reduction in the length of the G1 phase. A comparison of six cell lines revealed a linear relationship between the intracellular CaM concentration and the rate of G1 progression. These data provide the first evidence that specific elevation of CaM levels directly affects the rate of cell proliferation.

Animals↗
PubMed1987

Proteolysis of smooth muscle myosin light chain kinase. Formation of inactive and calmodulin-independent fragments.

Proteolysis by trypsin of gizzard myosin light chain kinase in the absence of Ca2+-calmodulin causes a biphasic effect on kinase activity. During the initial phase of proteolysis, Ca2+-calmodulin-dependent kinase activity is reduced over a thousand-fold. Further proteolysis, in the second phase, causes an increase in activity that is independent of Ca2+-calmodulin. Loss of activity is associated with the formation of a 64,000-dalton fragment. Calmodulin-independent activity is associated with the formation of a 61,000-dalton fragment. Procedures for the isolation of each fragment are outlined. Tryptic hydrolysis of the isolated 64,000-dalton peptide generates the 61,000-dalton peptide and increases calmodulin-independent activity. Km values for ATP and light chains for the native kinase and two fragments are the same, i.e. approximately 100 and 5 microM, respectively. Neither fragment binds to F-actin. Amino acid analyses of both fragments are given. Synthetic peptides corresponding to the calmodulin-binding regions of the smooth and skeletal muscle kinases are potent inhibitors of the 61,000-dalton fragment. These data demonstrate the existence of an inhibitory region that is suggested to be located between the active site and the calmodulin-binding site. Whether it is distinct from or at the N-terminal end of the calmodulin-binding site cannot be determined from these data.

Amino Acid Sequence↗
PubMed1987

Functional analysis of a complementary DNA for the 50-kilodalton subunit of calmodulin kinase II.

The calcium-calmodulin-dependent protein kinase II is a major component of brain synaptic junctions and has been proposed to play a variety of important roles in brain function. A complementary DNA representing a portion of the smaller 50-kilodalton subunit of the rat brain enzyme has been cloned and sequenced. The calmodulin-binding region has been identified and a synthetic analog prepared that binds calmodulin with high affinity in the presence of calcium. Like the 50-kilodalton kinase polypeptide, the concentration of the messenger RNA varies both neuroanatomically and during postnatal development of the brain. The broad tissue and species cross-reactivity of the complementary DNA suggests that the 50-kilodalton subunit found in rat brain is evolutionarily conserved and is the product of a single gene.

Age Factors↗
PubMed1987

Structural organization and chromosomal assignment of the parvalbumin gene.

The structure of the rat parvalbumin gene has been elucidated from analysis of six overlapping clones isolated from a rat lambda Charon 4A genomic library. Two of the clones were mapped in detail, and all exons were localized by Southern hybridization using fragments of a full-length parvalbumin cDNA (Epstein, P., Means, A. R., and Berchtold, M. W. (1986) J. Biol. Chem. 261, 5886-5891). The rat parvalbumin transcription unit is 15.5 kilobase pairs in length and contains four introns. The first intron divides the 5'-nontranslated region, whereas the other three interrupt coding DNA. All intron/extron boundaries were sequenced as was 377 base pairs immediately 5' from the putative transcription initiation site. The promoter region contains eukaryotic regulatory homologies to the "TATA" box at -24 and "CAAT" box at -47 and -156. In addition, two doublets consisting of 11-base pair direct repeats exist in the promoter region. Parvalbumin binds two Ca2+, whereas many other members of the same superfamily bind four. Comparison of the genes that encode these proteins provides a strong confirmation of the hypothesis that parvalbumin evolved from an ancestral gene specifying a four-domain Ca2+-binding protein. The rat parvalbumin gene was also utilized to assign its human counterpart to chromosome 22 from data obtained by hybridization to DNA from a somatic cell hybrid panel. It was also used to isolate a 7.5-kilobase pair EcoRI fragment from a human chromosome 22 DNA library.

Amino Acid Sequence↗
PubMed1987

Bacterially synthesized vertebrate calmodulin is a specific substrate for ubiquitination.

Calmodulin purified from bacteria which express a cloned chicken calmodulin gene can be selectively conjugated with ubiquitin, using enzymes present in reticulocyte extracts. Analyses of peptide products generated from limited proteolytic digestion of the calmodulin conjugate containing a single ubiquitin indicate that lysine 115 on calmodulin is the site of linkage. This linkage site is identical to that previously reported for calmodulin purified from Dictyostelium discoideum. Substrate-dependent ATP hydrolysis by a partially purified ubiquitin conjugation enzyme system from reticulocyte extracts was used to determine the enzyme affinity to calmodulin. Km values of 7 and 9 microM were determined for dictyostelium and the bacterially expressed calmodulin, respectively. The bacterially expressed calmodulin, unlike the Dictyostelium protein, can also form conjugates containing a 2-5 molar ratio of ubiquitin but at a slower rate than that observed for conjugation at lysine 115. Results from these studies further support our hypothesis that the post-translational methylation of lysine 115 found in most forms of calmodulin serves the important function of protecting calmodulin from ubiquitination and from degradation by the cytoplasmic ubiquitin-dependent proteolytic pathway. The capability of the bacterially expressed calmodulin to form conjugates with a high molar ratio of ubiquitin suggests that the post-translational acetylation of the N terminus of calmodulin may serve a similar function.

Adenosine Triphosphate↗
PubMed1987

The calmodulin binding domain of chicken smooth muscle myosin light chain kinase contains a pseudosubstrate sequence.

Smooth muscle myosin light chain kinase contains a 64 residue sequence that binds calmodulin in a Ca2+-dependent manner (Guerriero, V., Jr., Russo, M. A., and Means, A. R. (1987) Biochemistry, in press). Within this region is a sequence with homology to the corresponding sequence reported for the calmodulin binding region of skeletal muscle myosin light chain kinase (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, L., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3187-3191). Inspection of these sequences reveals that they both share a similar number and spatial arrangement of basic residues with those present in the myosin light chain substrate. We have synthesized a 22-residue peptide corresponding to residues 480-501 (determined from the cDNA) of the smooth muscle myosin light chain kinase. This peptide, Ala-Lys-Lys-Leu-Ser-Lys-Asp-Arg-Met-Lys-Lys-Tyr-Met-Ala-Arg-Arg-Lys-Trp- Gln-Lys-Thr-Gly, inhibited calmodulin-dependent activation of the smooth muscle myosin light chain kinase with an IC50 of 46 nM. At saturating concentrations of calmodulin, the 22-residue peptide inhibited myosin light chain and synthetic peptide substrate phosphorylation competitively with IC50 values of 2.7 and 0.9 microM, respectively. An 11-residue synthetic peptide analog, corresponding to part of the calmodulin-binding sequence in skeletal muscle myosin light chain kinase, Lys-Arg-Arg-Trp-Lys-Lys-Asn-Phe-Ile-Ala-Val, also competitively inhibited synthetic peptide substrate phosphorylation with a Ki of 1 microM. The competitive inhibitory activity of the calmodulin binding regions is similar to the apparent Km of 2.7 microM for phosphorylation of the 23-residue peptide analog of the smooth muscle myosin light chain and raises the possibility that the calmodulin binding region of the myosin light chain kinase may act as a pseudosubstrate inhibitor of the enzyme.

Amino Acid Sequence↗
PubMed1987

Association of calmodulin and smooth muscle myosin light chain kinase: application of a label selection technique with trace acetylated calmodulin.

A method is described for rapidly surveying the effects of modifying individual amino acid residues of a protein on its ability to interact specifically with another macromolecule. The procedure has been used to examine the individual roles of the seven lysyl residues of calmodulin in its ability to bind to smooth muscle myosin light chain kinase; previous studies by Jackson et al. (J. Biol. Chem. 261:1226-12232, 1986) have suggested that certain lysines may be located close to the interaction site. Trace [3H]-acetylated calmodulin, consisting predominantly of molecules acetylated at single sites together with unmodified protein, was incubated in excess (five- to 20-fold) with smooth muscle MLC kinase to allow the modified and unmodified molecules to compete for binding to the enzyme. Subsequently, the calmodulin-enzyme complex was separated from unbound calmodulin, and the level of acetylation of each of the seven lysines of the bound fraction of calmodulin was determined and compared to that of each corresponding group of the starting preparation. Significant changes were found at only two of the lysines, 21 and 75, where the extent of acetylation in the bound fraction was three- and fivefold lower, respectively, than that in the original preparation. These results were reproducible in three separate selection experiments employing both chicken and turkey gizzard MLC kinase. It is concluded that acetylation of calmodulin at either lysine 21 or 75 markedly reduces its affinity for MLC kinase, but acetylation at any of the other lysines (13, 30, 77, 94, or 148) has only minor effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗