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A R Means

Publications and source records attributed to A R Means.

At least 73 records · Page 4Linked to original sources
PubMed1994

Calcium/calmodulin-dependent protein kinase types II and IV differentially regulate CREB-dependent gene expression.

Phosphorylation of CREB (cyclic AMP [cAMP]- response element [CRE]-binding protein) by cAMP-dependent protein kinase (PKA) leads to the activation of many promoters containing CREs. In neurons and other cell types, CREB phosphorylation and activation of CRE-containing promoters can occur in response to elevated intracellular Ca2+. In cultured cells that normally lack this Ca2+ responsiveness, we confer Ca(2+)-mediated activation of a CRE-containing promoter by introducing an expression vector for Ca2+/calmodulin-dependent protein kinase type IV (CaMKIV). Activation could also be mediated directly by a constitutively active form of CaMKIV which is Ca2+ independent. The CaMKIV-mediated gene induction requires the activity of CREB/ATF family members but is independent of PKA activity. In contrast, transient expression of either a constitutively active or wild-type Ca2+/calmodulin-dependent protein kinase type II (CaMKII) fails to mediate the transactivation of the same CRE-containing reporter gene. Examination of the subcellular distribution of transiently expressed CaMKIV and CaMKII reveals that only CaMKIV enters the nucleus. Our results demonstrate that CaMKIV, which is expressed in neuronal, reproductive, and lymphoid tissues, may act as a mediator of Ca(2+)-dependent gene induction.

Base Sequence↗
PubMed1993

Biochemical characterization of the multifunctional Ca2+/calmodulin-dependent protein kinase type IV expressed in insect cells.

We have expressed the rat brain Ca2+/calmodulin (CaM)-dependent protein kinase type IV in insect cells. The recombinant enzyme is produced as a single polypeptide that migrates on SDS-polyacrylamide gel electrophoresis at 61 kDa. Recombinant CaM kinase IV undergoes slow CaM-dependent autophosphorylation. The autophosphorylation of CaM kinase IV occurs on serine residues but is not accompanied by the generation of a CaM-independent activity, as previously reported for the cerebellar enzyme. Comparison of peptide and protein phosphorylation by the recombinant CaM kinase IV and the cerebellar enzyme showed differences in their catalytic activities. The deduced primary sequence of CaM kinase IV contained a domain, 315Phe-Asn-Ala-Arg-Arg-Lys-Leu-Lys323, also found in the regulatory domain of CaM kinase II alpha (residues 293-300). Truncation of CaM kinase IV at Leu313 (at a position analogous to Leu290 in CaM kinase II alpha) generated a fully active, CaM-independent enzyme. This truncated enzyme no longer bound CaM. These data confirm that CaM kinase IV demonstrates intrasteric regulation by an autoinhibitory domain and provides insight into a potentially common mechanism for the regulation of the CaM-dependent multifunctional protein kinases. A number of synthetic peptides were examined for their phosphorylation by both CaM kinase II and IV. These studies showed that several peptides derived from phospholamban were preferential substrates for CaM kinase II whereas a peptide derived from S6 ribosomal protein was selectively phosphorylated by CaM kinase IV. Kinetic analysis of several peptide substrates suggests that while both CaM kinase II and IV recognize the sequence motif represented by R-X-X-T/S, other structural features are also involved in defining the unique substrate specificity of CaM kinase IV.

Amino Acid Sequence↗
PubMed1993

Modulation of calmodulin plasticity in molecular recognition on the basis of x-ray structures.

Calmodulin is the primary calcium-dependent signal transducer and regulator of a wide variety of essential cellular functions. The structure of calcium-calmodulin bound to the peptide corresponding to the calmodulin-binding domain of brain calmodulin-dependent protein kinase II alpha was determined to 2 angstrom resolution. A comparison to two other calcium-calmodulin structures reveals how the central helix unwinds in order to position the two domains optimally in the recognition of different target enzymes and clarifies the role of calcium in maintaining recognition-competent domain structures.

Amino Acid Sequence↗
PubMed1993

Calmodulin-cardiac troponin C chimeras. Effects of domain exchange on calcium binding and enzyme activation.

Calmodulin (CaM) and the cardiac isoform of troponin C (cTnC) are close structural homologs, but cTnC cannot activate most CaM target enzymes. To investigate structure-function relationships, we constructed a series of CaM.cTnC chimeras and determined their ability to bind Ca2+ and activate CaM target enzymes. Previously, we exchanged domain 1 and found that the chimeras exhibited profoundly impaired activation of smooth muscle myosin light chain kinase (smMLCK) and had differential effects on other CaM target enzymes (George, S. E., VanBerkum, M. F. A., Ono, T., Cook, R., Hanley, R. M., Putkey, J. A., and Means, A. R. (1990) J. Biol. Chem. 265, 9228-9235). One of the domain 1 chimeras was a potent competitive inhibitor of smMLCK. We now extend our study of CaM.cTnC chimeras by exchanging all of the remaining functional domains of CaM and cTnC. We determined the ability of the chimeras to bind Ca2+ and activate phosphodiesterase (PDE) and smMLCK. Chimeras containing both domains 3 and 4 of cTnC exhibited high affinity Ca2+ binding that was indistinguishable from cTnC, whereas chimeras containing either domain 3 or 4 of cTnC demonstrated Ca2+ affinity that was intermediate between CaM and cTnC. All of the CaM.cTnC chimeras showed near-maximal PDE activation but required 5-775-fold higher concentrations than CaM to produce half-maximal PDE activation. In contrast, all of the chimeras showed impaired ability to activate smMLCK, and some were potent competitive inhibitors of smMLCK activation by CaM.

Amino Acid Sequence↗
PubMed1993

Activation of four enzymes by two series of calmodulin mutants with point mutations in individual Ca2+ binding sites.

Activation of four target enzymes by two series of calmodulin Ca2+ binding site mutants has been examined. In each mutant, the conserved bidentate glutamate of one of the Ca2+ binding sites is mutated to glutamine or lysine. The enzymes studied were smooth and skeletal muscle myosin light chain kinases, adenylylcyclase, and plasma membrane Ca(2+)-ATPase. For the first three enzymes, the activation patterns with the two mutant series were very similar: mutation of site 4 was most deleterious, then site 2, site 3, and site 1. This ranking was observed previously in Ca2+ binding and Ca(2+)-induced conformational studies of these mutants. Thus the response of these enzymes is probably determined by the extent to which each mutant's competence to interact with target binding regions has been compromised. In contrast, for Ca(2+)-ATPase, mutants of sites 3 and 4 were much poorer activators than those of sites 1 and 2. Events beyond calmodulin binding and related to enzyme activation probably dictate this unusual activation pattern and also the anomalously poor activation of skeletal muscle myosin light chain kinase by site 1 mutant B1Q. Site 1 mutant B1K showed wild type activation of all four enzymes suggesting that in site 1, the lysine substitution can evoke the conformational changes associated with Ca2+ binding.

Adenylyl Cyclases↗
PubMed1993

Properties and regulation of the cell cycle-specific NIMA protein kinase of Aspergillus nidulans.

NIMA is the protein product of the nimA gene of the filamentous fungus Aspergillus nidulans, required for progression of cells from G2 into mitosis. The protein kinase activity of NIMA, assayed by phosphorylation of beta-casein, varies during the nuclear division cycle, reaching a maximum in late G2 and M. To investigate the biochemical properties of this cell cycle-regulated protein kinase, we have expressed nimA cDNA that encodes full-length NIMA in Escherichia coli as a fusion product with glutathione S-transferase. Purified NIMA phosphorylated beta-casein, with a Km of 38 microM and Vmax of 156 nmol/min/mg. NIMA also demonstrated a Km of 69 microM for ATP. Both recombinant and cellular NIMA kinases behaved as oligomers on gel filtration chromatography, and their kinase activities were strongly inhibited by various salts. By using both protein and peptide substrates, NIMA demonstrated a serine/threonine-specific protein kinase activity. Cellular NIMA exists as a phosphoprotein, and bacterially expressed NIMA was also phosphorylated on multiple serine/threonine residues. Some of these phosphorylations appeared essential for NIMA activity as the enzyme could be dephosphorylated and inactivated in vitro by protein serine/threonine phosphatases. Use of a kinase-negative mutant of NIMA revealed that the NIMA enzyme undergoes autophosphorylation when expressed at high concentrations in bacteria. Taken together, these data suggest that cellular mechanisms may exist to regulate the phosphorylation state and activity of the NIMA protein kinase during the nuclear division cycle in A. nidulans.

Aspergillus nidulans↗
PubMed1993

Coordinate regulation of mRNAs from multiple calmodulin genes during myoblast differentiation in vitro.

Multiple genes encoding identical calmodulin molecules have been found in all mammalian species so far examined, but little is known regarding the factors involved in regulating the expression of this gene family. We have investigated the possibility of differential regulation under conditions of cell cycle withdrawal and differentiation in the nonfusing BC3H1 myoblast. Transcripts from the three genes are expressed in myoblasts and myocytes and each of the mRNA species decreases during BC3H1 differentiation. Calmodulin protein levels also decrease, although with distinct kinetics with respect to the mRNAs. Previous studies indicated that a decrease in transcription is involved (Epstein et al., Molecular Endocrinology 3:193-202, 1989). In this study, an increase in stability for each of the mRNA species is also shown to contribute to overall mRNA levels. The calmodulin mRNAs are also found to decrease under conditions of cell cycle withdrawal when differentiation is blocked. This demonstrates that the expression of mRNA from all three genes is directly coupled with the proliferation state but only indirectly with the differentiation state. Consistent with this, calmodulin expression decreases in serum deprived fibroblasts as they exit the cell cycle.

Animals↗
PubMed1993

Essential roles for calcium and calmodulin in G2/M progression in Aspergillus nidulans.

nimT encodes a protein in Aspergillus nidulans that is required for tyrosine dephosphorylation of p34cdc2 and has a strong homology to cdc25-type proteins. Conditional mutation of nimT (nimT23 mutation) arrests cells in G2 at the restrictive temperature. After release of the temperature-sensitive nimT23 block, p34cdc2 undergoes tyrosine dephosphorylation and we showed that as cells entered mitosis, a rapid increase in calmodulin was observed. The increase in calmodulin and progression into mitosis were prevented by reducing extracellular Ca2+ levels to 2 nM. The calmodulin gene of a nimT23-containing strain was replaced with a hybrid gene in which calmodulin transcription was regulated by the alcA promoter (AlcCaM/T23). This allowed experimental manipulation of the level of intracellular calmodulin by the carbon source in the medium. When either extracellular Ca2+ or intracellular calmodulin levels were reduced at the nimT23 G2 arrest point, p34cdc2 remained tyrosine phosphorylated but the mitotic NIMA kinase encoded by nimA was not activated. Release of the temperature sensitive nimT23 arrest when either extracellular Ca2+ or calmodulin concentrations were low blocked tyrosine dephosphorylation of p34cdc2, activation of NIMA and progression of cells into mitosis. However, reduced levels of either Ca2+ or calmodulin had no effect on the increase in histone H1 kinase activity associated with p13 beads or the degree of phosphorylation of the majority of MPM-2-reacting proteins following release of the nimT23 mutation. These results demonstrate that both Ca2+ and calmodulin are important for progression into mitosis from the nimT23 arrest point in a pathway involving activation of both NIMA and p34cdc2 protein kinases.

Aspergillus nidulans↗
PubMed1993

Targeted developmental overexpression of calmodulin induces proliferative and hypertrophic growth of cardiomyocytes in transgenic mice.

Calmodulin (CaM) levels are developmentally regulated in the mouse heart. During late gestational and early postnatal stages, CaM levels decline several-fold in close temporal association with the declining population of proliferating cardiomyocytes. This correlation suggests that CaM may influence cardiomyocyte cell cycle activity, particularly since CaM is implicated in cell cycle control in several eukaryotic nonmuscle cells. To test this possibility, nucleotides -500 to 77 of the human atrial natriuretic factor gene were linked to a chicken CaM minigene to establish two pedigrees of transgenic mice that express 3- to 5-fold increased levels of CaM in cardiomyocytes. Developmental overexpression of CaM in mouse cardiomyocytes produced a markedly exaggerated cardiac growth response, characterized by the presence of cardiomyocyte hypertrophy in regions demonstrated to overexpress CaM and by cardiomyocyte hyperplasia, apparent at early developmental stages. Early postnatal suppression of fusion gene expression in the cardiac ventricles correlated with regression of the ventricular growth response in transgenic relative to nontransgenic mice between 3 days and 6-10 weeks of age, but was not apparent in the cardiac atria, where levels of CaM remained constitutively elevated until advanced stages. To test the possibility that increased cytosolic Ca2+ buffering contributes to the growth response induced by CaM over-expression, two additional lines of transgenic mice were generated using the same human atrial natriuretic factor promoter to target expression of a CaM mutant (amino acids 75-82 deleted) in cardiomyocytes. This mutant has previously been shown to bind Ca2+ with kinetic properties similar to those of wild-type CaM, but was unable to activate several CaM-dependent target enzymes in vitro. Despite high level expression of the CaM mutant, no growth response was apparent in the hearts of transgenic relative to those of nontransgenic mice, suggesting that increased Ca2+ buffering is unlikely to contribute to the growth response induced by CaM overexpression. Taken together, these findings reveal that cardiomyocyte growth regulation is specifically influenced by CaM concentrations in transgenic mice.

Animals↗
PubMed1992

Calmodulin structure refined at 1.7 A resolution.

We have determined and refined the crystal structure of a recombinant calmodulin at 1.7 A resolution. The structure was determined by molecular replacement, using the 2.2 A published native bovine brain structure as the starting model. The final crystallographic R-factor, using 14,469 reflections in the 10.0 to 1.7 A range with structure factors exceeding 0.5 sigma, is 0.216. Bond lengths and bond angle distances have root-mean-square deviations from ideal values of 0.009 A and 0.032 A, respectively. The final model consists of 1279 non-hydrogen atoms, including four calcium ions, 1130 protein atoms, including three Asp118 side-chain atoms in double conformation, 139 water molecules and one ethanol molecule. The electron densities for residues 1 to 4 and 148 of calmodulin are poorly defined, and not included in our model, except for main-chain atoms of residue 4. The calmodulin structure from our crystals is very similar to the earlier 2.2 A structure described by Babu and coworkers with a root-mean-square deviation of 0.36 A. Calmodulin remains a dumb-bell-shaped molecule, with similar lobes and connected by a central alpha-helix. Each lobe contains three alpha-helices and two Ca2+ binding EF hand loops, with a short antiparallel beta-sheet between adjacent EF hand loops and one non-EF hand loop. There are some differences in the structure of the central helix. The crystal packing is extensively studied, and facile crystal growth along the z-axis of the triclinic crystals is explained. Herein, we describe hydrogen bonding in the various secondary structure elements and hydration of calmodulin.

Calcium↗
PubMed1992

Regulation of intrasteric inhibition of the multifunctional calcium/calmodulin-dependent protein kinase.

A regulatory region involved in both autoinhibition and calmodulin (CaM) binding has previously been identified in the multifunctional Ca2+/CaM-dependent protein kinase (CaM kinase II). We have tested the role of various segments of the regulatory region in autoinhibition by the analysis of a series of truncation, substitution, and deletion mutants of the CaM kinase II alpha subunit (CaM kinase II alpha). Unexpectedly, the sequence Lys-Lys-Phe-Asn at positions 291-294, adjacent to the CaM binding domain, was found to be sufficient to maintain an inhibited state in a truncated form of the kinase. However, these residues are not essential in the context of the full-length protein, indicating the importance of additional residues from the overlapping CaM binding domain. We propose here a molecular model for CaM kinase II alpha based on the three-dimensional structure of the cAPK-PKI-(5-24) (protein kinase inhibitor fragment) complex. It is predicted from this model that autoinhibition is of the pseudosubstrate variety and that autophosphorylation of Thr-286 could occur by an intersubunit reaction in the holoenzyme complex.

Allosteric Regulation↗
PubMed1992

Structural basis of the intrasteric regulation of myosin light chain kinases.

The smooth muscle myosin light chain kinase (smMLCK) catalytic core was modeled by using the crystallographic coordinates of the cyclic AMP-dependent protein kinase catalytic subunit (cAPK) and a bound pseudosubstrate inhibitor peptide, PKI(5-24). Despite only 30% identity in amino acid sequence, the MLCK sequence can be readily accommodated in this structure. With the exception of the short B-helix, all major elements of secondary structure in the core are very likely conserved. The active site of the modeled MLCK complements the known requirements for peptide substrate recognition. MLCK contains a pseudosubstrate sequence that overlaps the calmodulin binding domain and has been proposed to act as an intrasteric inhibitor and occupy the substrate binding site in the absence of Ca(2+)-calmodulin. The pseudosubstrate sequence can be modeled easily into the entire backbone of PKI(5-24). The results demonstrate that the intrasteric model for regulation of MLCK by intramolecular competitive inhibition is structurally plausible.

Amino Acid Sequence↗
PubMed1992

Target enzyme recognition by calmodulin: 2.4 A structure of a calmodulin-peptide complex.

The crystal structure of calcium-bound calmodulin (Ca(2+)-CaM) bound to a peptide analog of the CaM-binding region of chicken smooth muscle myosin light chain kinase has been determined and refined to a resolution of 2.4 angstroms (A). The structure is compact and has the shape of an ellipsoid (axial ratio approximately 2:1). The bound CaM forms a tunnel diagonal to its long axis that engulfs the helical peptide, with the hydrophobic regions of CaM melded into a single area that closely covers the hydrophobic side of the peptide. There is a remarkably high pseudo-twofold symmetry between the closely associated domains. The central helix of the native CaM is unwound and expanded into a bend between residues 73 and 77. About 185 contacts (less than 4 A) are formed between CaM and the peptide, with van der Waals contacts comprising approximately 80% of this total.

Amino Acid Sequence↗
PubMed1992

Identification of amino acids essential for calmodulin binding and activation of smooth muscle myosin light chain kinase.

Smooth muscle myosin light chain kinase (smMLCK) is a Ca(2+)-calmodulin (CaM)-dependent enzyme that phosphorylates the 20-kDa light chains of myosin. In a previous study (Bagchi, I.C., Kemp, B.E., and Means, A.R. (1989) J. Biol. Chem. 264, 15843-15849), we expressed in bacteria a 40-kDa fragment of smMLCK that displayed Ca(2+)-CaM-regulated catalytic activity. Initial mutagenesis experiments indicated that Gly811 and Arg812 were important for CaM-dependent activation of this 40-kDa enzyme. We have now carried out site-directed mutagenesis within the CaM-binding domain (Ser787 to Leu813) of this enzyme to identify amino acids that are critical for CaM binding and activation. Our studies reveal that the individual mutation of several hydrophobic amino acid residues such as Leu813, Ile810, and Trp800 and the glycine residue Gly804 also resulted in a severe decrease in or complete loss of CaM binding and activation of smMLCK. The hydrophobic residue (Trp800) and the basic residue (Arg812), both of which are mandatory for CaM binding to smMLCK, occur in analogous positions within the CaM-binding domain of a number of CaM-regulated enzymes. We conclude from these results that CaM binding by smMLCK is determined by an interplay of specific hydrophobic and electrostatic interactions which appear to be conserved among various target enzymes of CaM.

Amino Acid Sequence↗
PubMed1992

Characterization of the secondary structure of calmodulin in complex with a calmodulin-binding domain peptide.

The interaction between calcium-saturated chicken calmodulin and a peptide corresponding to the calmodulin-binding domain of the chicken smooth muscle myosin light chain kinase has been studied by multinuclear and multidimensional nuclear magnetic resonance methods. Extensive 1H and 15N resonance assignments of calmodulin in the complex have been obtained from the analysis of two- and three-dimensional nuclear magnetic resonance spectra. The assignment of calmodulin in the complex was facilitated by the use of selective labeling of the protein with alpha-15N-labeled valine, alanine, lysine, leucine, and glycine. These provided reference points during the main-chain-directed analysis of three-dimensional spectra of complexes prepared with uniformly 15N-labeled calmodulin. The pattern of nuclear Overhauser effects (NOE) seen among main-chain amide NH, C alpha H, and C beta H hydrogens indicates that the secondary structure of the globular domains of calmodulin in the complex closely corresponds to that observed in the calcium-saturated state of the protein in the absence of bound peptide. However, the backbone conformation of residues 76-84 adopts an extended chain conformation upon binding of the peptide in contrast to its helical conformation in the absence of peptide. A sufficient number of NOEs between the globular domains of calmodulin and the bound peptide have been found to indicate that the N- and C-terminal regions of the peptide interact with the C- and N-terminal domains of calmodulin, respectively. The significance of these results are discussed in terms of recently proposed models for the structure of calmodulin-peptide complexes.

Amino Acid Sequence↗
PubMed1992

Increased calmodulin affects cell morphology and mRNA levels of cytoskeletal protein genes.

We have previously described stable mouse C127 cell lines in which a CaM mini-gene has been expressed in a bovine papilloma virus-based expression vector (Rasmussen and Means: EMBO J. 6:3961-3968, 1987). Elevation of CaM to levels five-fold higher than in control cells caused an acceleration in cell cycle progression by reducing the length of the G1 period. When these cell lines were originally isolated it was observed that cells in which CaM levels were increased had a flattened morphology. In this study we have examined the localization of actin, vimentin, and tubulin in these cells as compared to the BPV-transformed control cell line in order to determine if changes in shape were accompanied by differences in the cytoskeletal organization. Cell-cycle-dependent changes in the levels of mRNAs for histone H4, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, vimentin, and beta-tubulin have also been examined. Our results indicate that increased CaM causes differences in the organization of microfilaments, intermediate filaments, and microtubules and that these changes are accompanied by selective differences in the cell-cycle-dependent expression of some mRNAs. Elevated CaM was also correlated with a reduced stability of beta-tubulin mRNA. These studies indicate that CaM has pleiotropic effects on cell function and suggest that stable cell lines with altered CaM levels may provide a useful model system for understanding the molecular basis of CaM-dependent regulation of cellular processes.

Actin Cytoskeleton↗