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A R Means

Publications and source records attributed to A R Means.

At least 55 records · Page 3Linked to original sources
PubMed1996

HER-2/c-erbB2 is phosphorylated by calmodulin-dependent protein kinase II on a single site in the cytoplasmic tail at threonine-1172.

Calmodulin-dependent protein kinase II (Cam kinase II) is known to desensitise epidermal growth factor receptor (HER-1) tyrosine kinase activity by a process involving phosphorylation at serines 1046/47 in the cytoplasmic tail. We have developed an experimental system to investigate phosphorylation of the related HER-2/c-erbB2 proto-oncogene utilising purified Cam kinase II and recombinant glutathione-S-transferase (GST) fusion proteins. The cDNA for rat Cam kinase II-alpha was transfected into human embryonic kidney (HEK) 293 fibroblasts and the expressed protein purified to homogeneity by calmodulin-agarose affinity chromatography. A GST fusion protein comprising residues 1126-1255 of HER-2 was phosphorylated by purified Cam kinase II, in contrast to a GST protein comprising residues 1005-1125. Phosphoamino-acid analysis and site-directed mutagenesis indicated that HER-2 was phosphorylated on a single site at threonine-1172 which resides within a consensus Cam kinase II phosphorylation site (RAKT). HER-2 (threonine-1172-alanine), in the form of a ligand-inducible chimaera HER-1/2, was co-transfected into HEK-293 fibroblasts with a constitutively active form of Cam kinase II, followed by in vivo labelling of these cells with 32 P-orthophosphate. Immunoprecipitation of ligand-activated receptors followed by two-dimensional phosphopeptide mapping indicated that threonine-1172 in HER-2 is a newly identified in vivo site which can be hyper-phosphorylated by constitutively active Cam kinase II. In addition, when over-expressed in HEK-293 fibroblasts, HER-1/2 (threonine-1172-alanine) showed a defect in desensitisation and underwent a more sustained EGF-induced receptor autophosphorylation compared to wild-type HER-1/2.

Amino Acid Sequence↗
PubMed1996

Multiple Ca(2+)-calmodulin-dependent protein kinase kinases from rat brain. Purification, regulation by Ca(2+)-calmodulin, and partial amino acid sequence.

We have purified to near homogeneity from rat brain two Ca(2+)-calmodulin-dependent protein kinase I (CaM kinase I) activating kinases, termed here CaM kinase I kinase-alpha and CaM kinase I kinase-beta (CaMKIK alpha and CaMKIK beta, respectively). Both CaMKIK alpha and CaMKIK beta are also capable of activating CaM kinase IV. Activation of CaM kinase I and CaM kinase IV occurs via phosphorylation of an equivalent Thr residue within the "activation loop" region of both kinases, Thr-177 and Thr-196, respectively. The activities of CaMKIK alpha and CaMKIK beta are themselves strongly stimulated by the presence of Ca(2+)-CaM, and both appear to be capable of Ca(2+)-CaM-dependent autophosphorylation. Automated microsequence analysis of the purified enzymes established that CaMKIK alpha and -beta are the products of distinct genes. In addition to rat, homologous nucleic acids corresponding to these CaM kinase kinases are present in humans and the nematode, Caenorhabditis elegans. CaMKIK alpha and CaMKIK beta are thus representatives of a family of enzymes, which may function as key intermediaries in Ca(2+)-CaM-driven signal transduction cascades in a wide variety of eukaryotic organisms.

Amino Acid Sequence↗
PubMed1996

Role of Ca++/calmodulin binding proteins in Aspergillus nidulans cell cycle regulation.

The goal of this review is to summarise the current knowledge concerning the targets of Ca++/calmodulin that are essential for cell cycle progression in lower eukaryotes. Emphasis is placed on Aspergillus nidulans since this is the only organism to date shown to posses essential Ca++ dependent calmodulin activated enzymes. Two such enzymes are the calmodulin activated protein phosphatase, calcineurin and the calmodulin dependent protein kinase. These proteins, each the product of a unique gene, are required for progression of quiescent spores into the proliferative cycle and also for execution of the nuclear division cycle in exponentially growing germlings.

Aspergillus nidulans↗
PubMed1996

Ca(2+)/calmodulin-dependent kinase is essential for both growth and nuclear division in Aspergillus nidulans.

The calmodulin gene has been shown to be essential for cell cycle progression in a number of eukaryotic organisms. In vertebrates and Aspergillus nidulans the calmodulin dependence also requires calcium. We demonstrate that the unique gene encoding a multifunctional calcium/calmodulin-dependent protein kinase (CaMK) is also essential in A. nidulans. This enzyme is required both for the nuclear division cycle and for hyphal growth, because spores containing the disrupted gene arrest with a single nucleus and fail to extend a germ tube. A strain conditional for the expression of CaMK was created. When grown under conditions that resulted in a 90% decrease in the enzyme, both nuclear division and growth were markedly slowed. The CaMK seems to be important for progression from G2 to mitosis.

Aspergillus nidulans↗
PubMed1995

Organization and analysis of the complete rat calmodulin-dependent protein kinase IV gene.

A 42-kilobase pair region of rat DNA containing the Ca2+/calmodulin-dependent protein kinase IV (CaM kinase IV) gene has been cloned and characterized. The gene consists of 12 exons and 11 introns and is predicted to encode both beta and alpha forms of CaM kinase IV as well as the testis-specific calmodulin-binding protein calspermin. The promoter utilized to generate the alpha-kinase isoform is located in intron 1, whereas the promoter utilized to produce the calspermin transcript is contained in intron 10. The calspermin promoter region which extends from -200 to +321 relative to the calspermin transcription initiation site that contains two cyclic AMP response elements (CRE) at -70 and -50 and has been shown previously to be inactive in NIH3T3 cells (Sun, Z., Sassone-Corsi, P., and Means, A. R. (1995) Mol. Cell. Biol. 15, 561-571) was ligated to the lacZ reporter gene and used to generate transgenic mice. The promoter was expressed exclusively in postmeiotic testis where beta-galactosidase was found predominantly in elongating spermatids. The cell and developmental specificity of transgene expression was very similar to the pattern shown by the endogenous gene. Although the transgene promoter was silent in somatic tissues, beta-galactosidase expression could be restored in primary cultures of skin fibroblasts by introduction of vectors encoding CREM tau and CaM kinase IV.

Amino Acid Sequence↗
PubMed1995

Defective glycolysis and calcium signaling underlie impaired insulin secretion in a transgenic mouse.

Pancreatic beta cells from mice that overexpress the Ca(2+)-binding protein calmodulin have a unique secretory defect that leads to chronic hyperglycemia. To further understand the molecular basis underlying this defect, we have studied signaling pathways in these beta cells. Measurements of cytosolic free Ca2+ concentration ([Ca2+]i) using fura-2 or indo-1 revealed a markedly reduced response when glucose was the stimulant. However, eliciting membrane depolarization with 50 mM K+ or the addition of the ATP-sensitive K+ (K+ ATP) channel antagonist tolbutamide restored [Ca2+]i transients to near normal levels. Electrophysiological analysis of the beta cell ion channels revealed that Ca2+ currents, delayed rectifier K+ currents, and K+ATP channel currents were similar in transgenic and nontransgenic cells, suggesting that these ion channels were able to function normally. However, whereas K+ATP channel currents in control cells were reduced by 50% by the presence of high glucose, those in transgenic cells were unaltered. Addition of tolbutamide inhibited this channel and enhanced the secretion of insulin in response to glucose for both control and transgenic cells. As these observations implicated a metabolic defect, glucose utilization, which is an indicator of glucose metabolism and ATP production in beta cells, was measured and found to be reduced by 40% in the transgenic cells. These data support the contention that excessive levels of calmodulin may compromise the ability of the beta cell to metabolize glucose and to modulate the state of the K+ATP channel, resulting in an inadequate control of the membrane potential, which collectively impair [Ca2+]i and thus insulin secretion in response to glucose.

Adenosine Triphosphate↗
PubMed1995

An intron facilitates activation of the calspermin gene by the testis-specific transcription factor CREM tau.

Calspermin is a high affinity Ca2+/calmodulin binding protein that is found only in postmeiotic male germ cells. Our previous studies have shown that the calspermin transcript is produced by utilization of a testis-specific promoter located within an intron of the calmodulin kinase IV gene. This promoter contains two cAMP response element-like motifs that bind the testis-specific transcription factor CREM tau. This interaction is required for transcriptional activation. Here we describe a novel regulatory element, the 111-base pair first intron of the calspermin gene, which is also required for enhancement of transcription by CREM tau via the cAMP response element motifs. Deletion or inversion of this intron results in loss of CREM tau-mediated stimulation of transcription. However, CREM tau stimulates calspermin promoter activity when the intron is moved upstream of the promoter but only when inserted in the proper orientation. Footprint, linker scanning, and deletion analyses were used to identify regulatory elements in the intron. We suggest that the intron functions as an orientation-dependent but position-independent regulatory element to activate the calspermin promoter by facilitating the stimulatory effect of CREM tau on transcription.

3T3 Cells↗
PubMed1995

Human calcium-calmodulin dependent protein kinase I: cDNA cloning, domain structure and activation by phosphorylation at threonine-177 by calcium-calmodulin dependent protein kinase I kinase.

Human Ca(2+)-calmodulin (CaM) dependent protein kinase I (CaMKI) encodes a 370 amino acid protein with a calculated M(r) of 41,337. The 1.5 kb CaMKI mRNA is expressed in many different human tissues and is the product of a single gene located on human chromosome 3. CaMKI 1-306, was unable to bind Ca(2+)-CaM and was completely inactive thereby defining an essential component of the CaM-binding domain to residues C-terminal to 306. CaMKI 1-294 did not bind CaM but was fully active in the absence of Ca(2+)-CaM, indicating that residues 295-306 are sufficient to maintain CaMKI in an auto-inhibited state. CaMKI was phosphorylated on Thr177 and its activity enhanced approximately 25-fold by CaMKI kinase in a Ca(2+)-CaM dependent manner. Replacement of Thr177 with Ala or Asp prevented both phosphorylation and activation by CaMKI kinase and the latter replacement also led to partial activation in the absence of CaMKI kinase. Whereas CaMKI 1-306 was unresponsive to CaMKI kinase, the 1-294 mutant was phosphorylated and activated by CaMKI kinase in both the presence and absence of Ca(2+)-CaM although at a faster rate in its presence. These results indicate that the auto-inhibitory domain in CaMKI gates, in a Ca(2+)-CaM dependent fashion, accessibility of both substrates to the substrate binding cleft and CaMKI kinase to Thr177. Additionally, CaMKI kinase responds directly to Ca(2+)-CaM with increased activity.

Amino Acid Sequence↗
PubMed1995

Phosphorylation and activation of Ca(2+)-calmodulin-dependent protein kinase IV by Ca(2+)-calmodulin-dependent protein kinase Ia kinase. Phosphorylation of threonine 196 is essential for activation.

Purified pig brain Ca(2+)-calmodulin (CaM)-dependent protein kinase Ia kinase (Lee, J. C., and Edelman, A. M. (1994) J. Biol. Chem. 269, 2158-2164) enhances, by up to 24-fold, the activity of recombinant CaM kinase IV in a reaction also requiring Ca(2+)-CaM and MgATP. The addition of brain extract, although capable of activating CaM kinase IV by itself, provides no further activation beyond that induced by purified CaM kinase Ia kinase, consistent with the lack of a requirement of additional components for activation. Activation is accompanied by the development of significant (38%) Ca(2+)-CaM-independent CaM kinase IV activity. In parallel fashion to its activation, CaM kinase IV is phosphorylated in a CaM kinase Ia kinase-, Ca(2+)-CaM-, and MgATP-dependent manner. Phosphorylation occurs on multiple serine and threonine residues with a Ser-P:Thr-P ratio of approximately 3:1. The identical requirements for phosphorylation and activation and a linear relationship between extent of phosphorylation of CaM kinase IV and its activation state indicate that CaM kinase IV activation is induced by its phosphorylation. Replacement of Thr-196 of CaM kinase IV with a nonphosphorylatable alanine by site-directed mutagenesis abolishes both the phosphorylation and activation of CaM kinase IV, demonstrating that Thr-196 phosphorylation is essential for activation.

Amino Acid Sequence↗
PubMed1995

Calspermin gene transcription is regulated by two cyclic AMP response elements contained in an alternative promoter in the calmodulin kinase IV gene.

The transcript for the high-affinity Ca2+/calmodulin-binding protein calspermin is generated from the gene encoding Ca2+/calmodulin-dependent protein kinase IV only in postmeiotic germ cells during spermatogenesis. We demonstrate that this testis-specific calspermin transcript can be produced in heterologous cells by utilization of a promoter located in an intron of the calmodulin (CaM) kinase IV gene. Critical motifs within this promoter are two cyclic AMP response element (CRE)-like sequences located about -70 and -50 bp upstream of the transcriptional initiation site. Both CRE motifs are footprinted by the authentic testis-specific transcriptional activator CREM tau or by CREM tau present in adult testis nuclear extract. Whereas a 2.1-kb DNA fragment containing the calspermin promoter is inactive when transfected into NIH 3T3 cells, activity can be restored by cotransfection of CREM tau and protein kinase A or CaM kinase IV but not CaM kinase II alpha. Restoration of activity is greatly reduced by mutation of the two CRE motifs. Since CRE-like motifs have been identified in many genes uniquely expressed in postmeiotic germ cells, which contain abundant CREM tau protein, we suggest that CREM tau may function as one transcription factor responsible for the expression of postmeiotic germ cell-specific genes.

3T3 Cells↗
PubMed1995

Targeted overexpression of an inactive calmodulin that binds Ca2+ to the mouse pancreatic beta-cell results in impaired secretion and chronic hyperglycemia.

We have previously reported that elevated levels of calmodulin in pancreatic beta-cells of mice resulted in a unique secretory defect. To determine if this effect was due to Ca2+ buffering, a mutant form of calmodulin that has an eight-amino acid deletion in the central helix (CaM-8) was used. The mutated calmodulin binds Ca2+ normally, but alters the ability to interact with known Ca2+/calmodulin-activated enzymes. In vitro competition analysis using HIT cell extracts verified that in the presence of Ca2+, CaM-8 exhibited at least a 100-fold lower affinity for calmodulin-binding proteins than did normal CaM in this model beta-cell. Transgenic mice were then generated by targeting the CaM-8 to pancreatic beta-cells. The CaM-8 mice were normoglycemic at birth, but developed a hyperglycemic condition starting at about 6 days of age. This condition was progressive and characterized by elevated blood glucose that coincided with reduced levels of pancreatic insulin and low circulating serum insulin levels. Hormone measurements and immunohistochemical analysis revealed that islets exhibited a nonimmune reduction of insulin immunoreactive beta-cells, reduced amounts of insulin, and a 5-fold higher level of CaM-8 protein relative to normal CaM protein. Perifusion assays were used to test the secretion response to glucose. CaM-8 islets demonstrated a reduction in first and second phase insulin secretion, which became progressively worse with age. Depolarization of the membrane with 50 mM K+ in the presence of high glucose did not significantly improve secretion. Carbachol, which is thought to act in beta-cells through the release of intracellular Ca2+ stores and activation of protein kinase-C, restored both phases of secretion to normal levels. These results suggest that disruption of intracellular Ca2+ homeostasis alone is sufficient to interfere with the insulin secretion pathway.

Animals↗
PubMed1994

Selective activation and inhibition of calmodulin-dependent enzymes by a calmodulin-like protein found in human epithelial cells.

A calmodulin-like protein, which is identical in size and 85% identical to vertebrate calmodulin, was recently identified by 'subtractive hybridization' comparison of transcripts expressed in normal versus transformed human mammary epithelial cells. Unlike the ubiquitous distribution of calmodulin, calmodulin-like protein expression is restricted to certain epithelial cells, and appears to be modulated during differentiation. In addition, calmodulin-like protein levels are often significantly reduced in malignant tumor cells as compared to corresponding normal epithelial cells. The current studies compare calmodulin-like protein functions with those of calmodulin. We find that calmodulin-like protein activation of multifunctional Ca2+/calmodulin-dependent protein kinase II (calmodulin kinase II) is equivalent to activation by calmodulin, but that four other calmodulin-dependent enzymes, cGMP phosphodiesterase, calcineurin, nitric-oxide synthase, and myosin-light-chain kinase, display much weaker activation by calmodulin-like protein than by calmodulin. In the case of myosin-light-chain kinase, calmodulin-like protein competitively inhibits calmodulin activation of the enzyme with a Ki value of 170 nM. Thus, calmodulin-like protein may have evolved to function as a specific agonist of certain calmodulin-dependent enzymes, and/or as a specific competitive antagonist of other calmodulin-dependent enzymes.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1994

The calmodulin-dependent protein phosphatase catalytic subunit (calcineurin A) is an essential gene in Aspergillus nidulans.

The gene encoding the homologue of the catalytic subunit of the Ca2+/calmodulin-regulated protein phosphatase 2B (calcineurin A) has been isolated from Aspergillus nidulans. This gene, cnaA+, is essential in this fungal system. Analysis of growth-arrested cells following gene disruption by homologous recombination reveals that they are blocked early in the cell cycle. The cnaA+ gene encodes a 2.5 kb mRNA and the deduced protein sequence is highly homologous to the calcineurin A subunit of other species. The mRNA varies in a cell cycle-dependent manner with maximal levels found early in G1 and considerably before the G1/S boundary. As calmodulin is also essential for A. nidulans cell cycle progression and levels rise before the G1/S boundary, our data suggest that calcineurin may represent a primary target for calmodulin at this cell cycle transition point.

Amino Acid Sequence↗
PubMed1994

Calcium, calmodulin and cell cycle regulation.

Calcium and its ubiquitous intracellular receptor calmodulin are required for cell proliferation. Studies in a variety of model systems are beginning to identify components of the calcium/calmodulin cascade required for movement of quiescent cells into the cell cycle as well as for proliferating cells to move from G1 to S, G2 to M and through mitosis. Two calcium/calmodulin-dependent enzymes, the multifunctional calcium/calmodulin-dependent protein kinase and the protein phosphatase 2B (calcineurin) as well as a spindle pole body protein that binds calmodulin in the absence of calcium have been shown to be essential at specific phases of the cell cycle. In addition, the status of the intracellular calcium pools is critical for normal traverse of the cell cycle.

Calcium↗
PubMed1994

The calmodulin-dependent protein phosphatase catalytic subunit (calcineurin A) is an essential gene in Aspergillus nidulans.

The gene encoding the homologue of the catalytic subunit of the Ca2+/calmodulin-regulated protein phosphatase 2B (calcineurin A) has been isolated from Aspergillus nidulans. This gene, cnaA+, is essential in this fungal system. Analysis of growth-arrested cells following gene disruption by homologous recombination reveals that they are blocked early in the cell cycle. The cnaA+ gene encodes a 2.5 kb mRNA and the deduced protein sequence is highly homologous to the calcineurin A subunit of other species. The mRNA varies in a cell cycle-dependent manner with maximal levels found early in G1 and considerably before the G1/S boundary. As calmodulin is also essential for A.nidulans cell cycle progression and levels rise before the G1/S boundary, our data suggest that calcineurin may represent a primary target for calmodulin at this cell cycle transition point.

Amino Acid Sequence↗
PubMed1994

Expression of the noncatalytic domain of the NIMA kinase causes a G2 arrest in Aspergillus nidulans.

Temperature-sensitive mutation of the nimA gene of Aspergillus nidulans causes a reversible G2 arrest, whereas overexpression of nimA causes premature entry into mitosis from which the cells cannot exit. The nimA gene encodes a Ser/Thr-specific protein kinase (NIMA) which contains an extended COOH-terminal noncatalytic domain. To evaluate the role of this enzyme in nuclear division control, we introduced various mutant nimA cDNAs under the control of the inducible alcohol dehydrogenase gene promoter into a strain of Aspergillus nidulans containing a temperature-sensitive nimA mutation (nimA5). While expression of the wild type NIMA complemented the nimA5 mutation and induced a premature mitotic arrest when overexpressed, expression of a kinase-negative NIMA containing a single amino acid mutation in the putative ATP-binding site could not rescue the nimA5 mutation but resulted in a specific G2 arrest when overexpressed. An identical phenotype was observed with cells expressing only the noncatalytic COOH-terminal domain of NIMA, whereas overexpression of the inactive kinase domain was without effect. The G2 arrest produced by overexpression of the full-length inactive or COOH-terminal NIMA molecules did not prevent activation of the endogenous NIMA or H1 kinase activity precipitable by p13 beads. We suggest that this dominant-negative phenotype results from competitive inhibition of the association of active NIMA with a cellular target(s) and that appropriate targeting is essential for the mitotic function of the NIMA kinase.

Alcohol Dehydrogenase↗
PubMed1994

Identification of substrate specificity determinants for the cell cycle-regulated NIMA protein kinase.

NIMA is a cell cycle-regulated protein kinase required for the G2/M transition in the filamentous fungus Aspergillus nidulans. Previous biochemical characterization of the recombinant enzyme indicated that NIMA is a protein serine/threonine specific kinase with beta-casein being the best substrate from the many proteins and peptides tested (Lu, K.P., Osmani, S.A., and Means, A.R. (1993) J. Biol. Chem. 268, 8769-8776). However, substrate specificity or physiologically relevant substrates for NIMA remained unknown. In search for a peptide substrate for this enzyme, we screened an assembled library of synthetic peptides that each contained a phosphorylation site for a known protein kinase and found an excellent peptide substrate for NIMA, phospholemman 42-72 (PLM(42-72)). NIMA kinase phosphorylated PLM(42-72) uniquely and stoichiometrically on Ser63 with a Vmax of 1.4 mumol/min/mg and apparent Km of 20.0 microM. These kinetic constants were about 10-fold higher and 3-fold lower than those for beta-casein, respectively. A detailed analysis of substrate specificity determinants using synthetic peptide analogs of PLM(42-72) indicated that Phe-Arg-Xaa-Ser/Thr represents the optimal primary sequence for NIMA kinase phosphorylation. Replacement of the Arg at P-2 with Ala resulted in a 6-fold increase in Km and 2-fold decrease in Vmax, while substitution of the Phe at P-3 with Ala abolished NIMA phosphorylation. These results reveal the unique nature of substrate recognition by the NIMA kinase and should prove valuable in the search for biologically relevant NIMA substrates.

Amino Acid Sequence↗