Bovine Brain Calcineurin Research Articles

Molecular cloning and biochemical characterization of bovine retina calcineurin

Calcineurin (CaN) is a member of ser/thr protein phosphatase family. Earlier, we have reported that CaN is present in all eye tissues, although the activity and protein expression varied (Seitz et al., Invest Opthalmol Vis Sci, 43:15-21, 2002). We have isolated a full-length cDNA encoding bovine retina CaN. The CaN A subunit consists of 511 amino acid residues. A 10 amino acid (ATVEAIEADE) deletion before the autoinhibitory domain was observed in bovine retina CaN A compared to bovine brain CaN A. The study on CaN activity and regulation demonstrated that different metal ions have different effects on its phosphatase activity. Ni(2+) was found to be the strongest stimulator, while Zn(2+) was found to inhibit CaN phosphatase activity. Mn(2+) was a relatively less effective stimulator compared to Ni(2+). Fe(2+) was also able to stimulate CaN phosphatase activity; in contrast, a previous study found Fe(2+) slightly inhibited CaN activity from bovine brain. The residues at 97-201 were found to be essential for bovine retina CaN A phosphatase activity. The residues at 407-456 also had an inhibitory effect on CaN A phosphatase activity in addition to the previously known autoinhibitory domain at 457-480. These observations suggest that bovine retina CaN A might possess some distinct structural characteristics.

Molecular cloning, expression, purification and characterization of calcineurin from bovine cardiac muscle

Calcineurin (CaN), also known as calmodulin-dependent phosphatase, was cloned from bovine cardiac muscle and the deduced amino acid sequences of CaN A revealed that it had an open reading frame of 511 amino acid residues. As compared to bovine brain CaN A, the cardiac enzyme contains a 10 amino acid (ATVEAIEADE) deletion before the autoinhibitory region. A deletion analysis of the catalytic domain revealed a 20% decrease in phosphatase activity when the N-terminal 200 amino acids were removed from CaN A as compared to the wild type enzyme. The C-terminal deletions of CaN A revealed that in addition to the autoinhibitory domain (residues 457–480), additional adjacent residues (407–456) also inhibited CaN activity. These results point to either a second autoinhibitory region within CaN A or an extension of the previously noted autoinhibitory region within the cardiac CaN A enzyme.

In vitro Proteolytic Degradation of Bovine Brain Calcineurin by m-Calpain

A major cause of neuronal dysfunction is due to altered Ca2+ regulation. An increase in Ca2+ influx can activate Ca2+-dependent enzymes including calpains, causing the proteolysis of its specific substrates. In the present study, calcineurin (CaN) was found to be proteolysed by a Ca2+-dependent cysteine protease, m-calpain. In the presence of Ca2+, the 60 kDa subunit (CaN A) was degraded to a 46 kDa immunoreactive fragment, whereas in the presence of Ca2+ /calmodulin (CaM) immunoreactive fragments of 48 and 54 kDa were observed. The beta-subunit (CaN B) was not proteolysed in either condition. The proteolysis of CaN A increased its phosphatase activity and rendered it totally CaM-independent after 10 min of proteolysis. The molecular weight of the proteolytic fragments suggested that the m-calpain cleaved CaN A in the CaN B binding domain. A CaM-overlay experiment revealed that the CaM-binding site was present only in the 54 kDa fragment produced by CaN A proteolysis in the presence of Ca2+ /CaM. Thus, the increase in CaN A phosphatase activity observed in many neuronal disorders, may be due to the action of calpain.

Response of recombinant calcineurin to metal ions, reduction–oxidation agents, and enzymatic modification

Recombinant calcineurin heterodimer with the full length δ-isoform of the catalytic subunit (CaN 500) was expressed in insect cells using the baculovirus system and compared to native bovine brain enzyme in its response to divalent metal ions, redox reagents, and enzymatic modification of arginine residues. The response to various metal ions showed essentially the same profile as bovine brain calcineurin, although Co 2+ and Zn 2+ did not support recombinant activity as well. Kinetic analysis showed that metal ion and substrate binding were not independent, as found for the bovine brain calcineurin. Incubation with DTT or ascorbate alone caused similar effects on the activity of both enzymes, but different responses were observed when incubated with both DTT and ascorbate; only the recombinant enzyme showed activation. Arginine deimination of recombinant calcineurin by peptidylarginine deiminase resulted in the loss of 60–80% of its phosphatase activity with protection observed if calmodulin was present. Recombinant calcineurin was reactivated by treatment with the protease clostripain, suggesting that deimination of an arginine in the carboxyl terminal domain may be responsible for the loss of phosphatase activity and decreased calmodulin binding [Arch. Biochem. Biophys. 318 (1995) 370]. Supporting this conclusion, a truncated variant of the catalytic subunit lacking the carboxyl terminus showed no loss of phosphatase activity compared to full length calcineurin subunit and contained lower amounts of citrulline than the full length subunit after deimination. These different responses of recombinant calcineurin are consistent with conformational differences compared to bovine brain calcineurin and raise questions about its utility for studying the mechanism of calcineurin.

Inactivation of calcineurin by hydrogen peroxide and phenylarsine oxide. Evidence for a dithiol-disulfide equilibrium and implications for redox regulation.

Calcineurin (CaN) is a Ca2+-and calmodulin (CaM)-dependent serine/threonine phosphatase containing a dinuclear Fe-Zn center in the active site. Recent studies have indicated that CaN is a possible candidate for redox regulation. The inactivation of bovine brain CaN and of the catalytic CaN A-subunit from Dictyostelium by the vicinal dithiol reagents phenylarsine oxide (PAO) and melarsen oxide (MEL) and by H2O2 was investigated. PAO and MEL inhibited CaN with an IC50 of 3-8 microM and the inactivation was reversed by 2, 3-dimercapto-1-propane sulfonic acid. The treatment of isolated CaN with hydrogen peroxide resulted in a concentration-dependent inactivation. Analysis of the free thiol content performed on the H2O2 inactivated enzyme demonstrated that only two or three of the 14 Cys residues in CaN are modified. The inactivation of CaN by H2O2 could be reversed with 1,4-dithiothreitol and with the dithiol oxidoreductase thioredoxin. We propose that a bridging of two closely spaced Cys residues in the catalytic CaN A-subunit by PAO/MEL or the oxidative formation of a disulfide bridge by H2O2 involving the same Cys residues causes the inactivation. Our data implicate a possible involvement of thioredoxin in the redox control of CaN activity under physiological conditions. The low temperature EPR spectrum of the native enzyme was consistent with a Fe3+-Zn2+ dinuclear centre. Upon H2O2-mediated inactivation of the enzyme no significant changes in the EPR spectrum were observed ruling out that Fe2+ is present in the active enzyme and that the dinuclear metal centre is the target for the oxidative inactivation of CaN.

Spectroscopic and Enzymatic Characterization of the Active Site Dinuclear Metal Center of Calcineurin: Implications for a Mechanistic Role

The active site of bovine brain calcineurin contains an Fe3+-Zn2+ dinuclear metal center. Replacement of Zn2+ with Fe2+ yields a mixed valence Fe3+-Fe2+ center that exhibits a characteristic EPR signal that can be used as a convenient spectroscopic probe of the active site. Addition of product phosphate to both the Fe3+-Fe2+ and Fe3+-Zn2+ forms of calcineurin led to perturbations of the respective EPR signals, indicating that phosphate affects the environment of the paramagnetic centers. Anaerobic titrations of the iron-substituted Fe3+-Fe2+ enzyme with dithionite resulted in a gradual loss of activity toward pNPP that paralleled the loss of intensity of the EPR signal of the mixed valence diiron center. During dithionite reduction, an EPR resonance with g approximately 12 appeared. The intensity of this resonance increased when the spectrum was recorded in a parallel mode cavity and was therefore attributed to a paramagnetic center with integer spin. Oxidation of the Fe3+-Fe2+ cluster to the diferric state by hydrogen peroxide also led to a loss of activity. These results indicate that the mixed valence oxidation state represents the catalytically competent form of the cluster. The dependence of the enzyme activity on the redox state of the cluster has implications for a mechanistic role.

Class II pyrethroids: Noninhibitors calcineurin

Type II pyrethroid insecticides have been reported to be potent inhibitors of bovine brain calcineurin (EC 3.1.3.16, Enan E and Matsumara F, Biochem Pharmacol 43: 1777–1784, 1992). In concentrations up to 10 −5 M, none of the pyrethroid insecticides used in this study caused inhibition of the calcineurin-dependent dephosphorylation of the 19-amino acid phosphopeptide derived from the regulatory subunit R-II of the cyclic adenosine 3″,5″-monophosphate (cAMP)-dependent protein kinase, which has been established as a good substrate for this enzyme. Neither did any of the compounds tested cause a shift in the inhibitory activity of okadaic acid (apparent K i of 5 μM). The assumption that calcineurin is generally inhibited by pyrethroid insecticides is incorrect, and the interpretation of cellular experiments in which this assumption has been made must be revised.

A novel, calcium-inhibitable casein kinase in Paramecium cells

This is the first identification of a Ca 2+-inhibitable casein kinase (CPK) which we have isolated from the 100 000× g supernatant of Paramecium cell homogenates. The 1000-fold enriched CPK activity depends on millimolar Mg 2+ and is inhibited by low concentrations of heparin or by ≥100 μM Ca 2+. Enzyme activity is stimulated by polylysine or polyarginine with either casein or with specific casein kinase-2 (CK-2) peptide substrates (RRRDDDSDDD and RREEETEEE). The enzymic properties are similar with GTP instead of ATP. CPK does not undergo autophosphorylation. In gel kinase assays, enzyme activity is associated with a 36 kDa band. Calmodulin as another characteristic substrate for mammalian CK-2 has not been phosphorylated by this protein kinase. Besides casein, CPK phosphorylates in vitro the catalytic subunit of bovine brain calcineurin (CaN), a typical substrate of type 1 mammalian casein kinase (CK-1) in vitro. Again this phosphorylation is significantly reduced by Ca 2+. Thus, CPK combines aspects of different casein kinases, but it is clearly different from any type known by its Ca 2+ inhibition. Since CPK also phosphorylates the exocytosis-sensitive phosphoprotein, PP63, in Paramecium, which is known to be dephosphorylated by CaN, an antagonistic Ca 2+-effect during phosphorylation/dephosphorylation cycles may be relevant for exocytosis regulation.

Calcium regulation of calcineurin phosphatase activity by its B subunit and calmodulin. Role of the autoinhibitory domain.

Calcineurin (CaN) contains an autoinhibitory element (residues 457-482) 43 residues COOH-terminal of the calmodulin-binding domain (Hashimoto, Y., Perrino, B. A., and Soderling, T. R. (1990) J. Biol. Chem. 265, 1924-1927) that regulates the Ca(2+)-dependent activation of its phosphatase activity. Substitution of Arg476 and Arg477 or Asp467 to Ala in the autoinhibitory peptide 457-482 significantly decreased its inhibitory potency. CaN A subunits with these residues mutated to Ala were coexpressed with the Ca(2+)-binding B subunit using the baculovirus/Sf9 cell system. Kinetic analysis showed that although the purified mutants had no activity in the absence of calcium, they were less dependent than the wild-type enzyme on calcium and calmodulin for activity. To determine if additional autoinhibitory motifs were present in the COOH terminus of calcineurin, the A subunit was truncated at residues 457 or 420 and co-expressed with B subunit. The Vmax values of both truncation mutants with or without Ca2+ were increased relative to wild-type calcineurin. The increased Ca(2+)-independent activity of CaN420 relative to CaN457 indicates the presence of additional autoinhibitory element(s) within residues 420-457. CaN420 had similar high Vmax values with or without Ca2+, but the Km value for peptide substrate was increased 5-fold to 125 microM in the absence of Ca2+. The Km values of all the expressed calcineurin species were increased in the absence of Ca2+. The CaN A or CaN A420 subunits alone have low Vmax and high Km (115 microM) values even in the presence of Ca2+. These results indicate that 1) there are several autoinhibitory motifs between the CaM-binding domain and the COOH terminus that are relieved by Ca2+ binding to CaM and the B subunit, 2) Ca2+ binding to the B subunit also regulates enzyme activity by lowering the Km of the catalytic subunit for substrate, 3) binding of the B subunit is required for high Vmax values even after removal of the autoinhibitory domain. These results are consistent with synergistic activation of calcineurin by Ca2+ acting through both CaM and the B subunit.

Mapping of the immunophilin-immunosuppressant site of interaction on calcineurin.

The interaction of the immunosuppressive complexes cyclosporin A-cyclophilin A and FK506 binding protein-FK506 with the Ca(2+)- and calmodulin-dependent protein phosphatase calcineurin has been investigated by means of photoaffinity labeling and chemical cross-linking. Photolabeling of purified bovine brain calcineurin with the affinity label [O-[4-[4-(1-diazo-2,2,2-trifluoroethyl)benzoyl]aminobutanoyl]-D- serine8]cyclosporin in the presence of cyclophilin A results, in addition to the labeling of cyclophilin itself, in the transfer of some of the chemical probe to both the catalytic subunit A and the regulatory subunit B of calcineurin. Chemical cross-linking studies with disuccinimidyl suberate in the presence of either cyclophilin A, B, or C in complex with cyclosporin A or FK506 binding protein-FK506 result on the other hand in the apparently exclusive and strictly immunosuppressant-dependent formation of covalent immunophilin-calcineurin B subunit products. Cross-linking of immunophilins to calcineurin B subunit requires the presence of subunit A. In the present study, using a set of recombinant maltose-binding protein fusion products representing different stretches of the catalytic subunit A, we were able to map the minimal calcineurin A sequence necessary for immunophilin-ligand-calcineurin B interaction to occur.

Activation of enzymes by calmodulins containing intramolecular cross-links

We have reacted calmodulins containing cysteines substituted at positions 3 and 146 or 5 and 146 with bismaleimidohexane (BMH) to generate intramolecularly cross-linked proteins termed BMHCM or BMHCM1, respectively. Reactions were also performed with N-ethylmaleimide (NEM) in place of BMH to generate corresponding S-ethylsuccinimidylated proteins termed NEMCM or NEMCM1. The abilities of these proteins to activate plant NAD kinase, erythrocyte Ca2±ATPase and bovine brain calcineurin activities were assessed. The BMH- or NEM-reacted proteins activate calcineurin activity as does control calmodulin. K act values for Ca2±ATPase activation by BMHCM and BMHCM1 are increased 10-fold relative to the control value, with no corresponding change in V max values. Activation of this enzyme by NEMCM or NEMCM1 is not different from the control. In NAD kinase activation experiments BMHCM and BMHCM1 are associated with a 10 to 20-foid increase in K act values and a 60–75% reduction in V max values relative to the control. NEMCM1 is not associated with any apparent changes in NAD kinase activation, however, NEMCM is associated with a 10-fold increase in the K act value. NEM-reacted calmodulin containing a cysteine only at position 3 is not associated with an increased K act value, implying that this change is due to interactions between S-(ethylsuccinimido)cysteines at positions 3 and 146. In conclusion, cross-linking and associated distortions in the structure of calmodulin appear to have little or no effect on activation of calcineurin enzyme activity. However, bending in the central helix and/or steric restrictions associated with cross-linking increase significantly the K act value for Ca2±ATPase and NAD kinase activation, and dramatically reduce maximal activation of NAD kinase activity.

Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform.

Calcineurin A was purified by calmodulin-Sepharose affinity chromatography from Sf9 cells infected with recombinant baculovirus containing the cDNA of a rat calcineurin A isoform. The Sf9-expressed calcineurin A has a low basal phosphatase activity in the presence of EDTA (0.9 nmol/min/mg) which is stimulated 3-5-fold by Mn2+. Calmodulin increased the Mn2+ stimulated activity 3-5-fold. Bovine brain calcineurin B increased the A subunit activity 10-15-fold, and calmodulin further stimulated the activity of reconstituted A and B subunits 10-15-fold (644 nmol/min/mg). The Km of calcineurin A for 32P-RII pep (a peptide substrate (DLDVPIPGRFDRRVSVAAE) for CaN), was 111 microM with or without calmodulin, and calmodulin increased the Vmax about 4-fold. The Km of reconstituted calcineurin A plus B for 32P-RII pep was 20 microM, and calmodulin increased the Vmax 18-fold without affecting the Km. CaN A467-492, a synthetic autoinhibitory peptide (ITSFEEAKGLDRINERMPPRRDAMP) from calcineurin, inhibited the Mn2+/calmodulin-stimulated activities of the reconstituted enzyme and the A subunit with IC50's of 25 microM and 90 microM, respectively. The reconstitution of the phosphatase activity of an expressed isoform of calcineurin A by purified B subunit and calmodulin may facilitate comparative studies of the regulation of calcineurin A activity by the B subunit and calmodulin.

Isolation and sequence of rat testis cdna for a calcium binding polypeptide similar to the regulatory subunit of calcineurin

We have cloned and sequenced rat testis cDNAs coding for a calcium binding polypeptide similar to calcineurin beta subunit, the Ca(2+)-binding subunit of the Ca2+/calmodulin stimulated protein phosphatase. Rat testis cDNA library was screened with a monoclonal antibody Va1 raised against bovine brain calcineurin beta subunit. The deduced amino acid sequence is similar to that of human brain calcineurin beta subunit with respect to containing four putative calcium binding sites. However, distinct differences were found: 1) The cloned cDNA had six amino acids polypeptide tail at carboxy-terminal which is absent in human brain calcineurin beta subunit. This amino acids tail makes the carboxy-terminal highly hydrophilic in contrast to the human brain beta subunit which is hydrophobic at carboxy-terminal; 2) eleven amino acids at the N terminal of the cloned cDNA were completely different from the corresponding region of the brain calcineurin beta subunit.

Identification of the calmodulin-regulated protein phosphatase, calcineurin, in rat pancreatic islets

The aim of this work was the identification of the calmodulin-stimulated protein phosphatase, calcineurin, in rat pancreatic islets. For this purpose, a high-affinity calcineurin antibody and the Western blotting technique were used to detect the presence of calcineurin in freshly collagenase-isolated islets. The calcineurin content detected by this method was about 0.30 ng islet (approx. 0.07% of the total islet protein). The subunit composition and Mr of islet calcineurin were similar to those of bovine brain calcineurin. Incubation of nitrocellulose membranes of the Western blotting, containing the islet protein fractions, with 125I-labeled calmodulin and 45Ca2+ demonstrated that the A subunit bound calmodulin, while the B subunit bound Ca2+. The presence of calcineurin in the islets of Langerhans would suggest its possible participation, as a counterpart of the kinases effect, in the regulatory mechanism of insulin secretion.

Divalent cation effects on calcineurin phosphatase: differential involvement of hydrophobic and metal binding domains in the regulation of the enzyme activity

The effects of divalent metals, metal chelators (EDTA, EGTA) and sodium dodecyl sulfate were investigated on the phosphatase activity of isolated bovine brain calcineurin assayed in the absence (called intrinsic) and presence of calmodulin. Intrinsic phosphatase was increased by Mn2+, was unaffected by Mg2+, Ca2+, and Ba2+, and was markedly inhibited by Ni2+, Fe2+, Zn2+ and Cu2+. When assayed in the presence of calmodulin, many divalent metals (Ni2+, Zn2+, Pb2+, Cd2+), besides Mn2+, increased modestly the phosphatase activity at low concentrations (10-100 microM) and inhibited it markedly at high concentrations. Ca2(+)-calmodulin stimulated phosphatase activity was antagonized by Ni2+, Zn2+, Fe2+, Cu2+, Pb2+, at low concentrations (50 microM), and by Ba2+, Cd2+ at slightly higher concentrations (greater than 100 microM); Mn2+ and Co2+ (50 microM to 1 mM) in fact augmented it. EDTA and EGTA in a concentration and time dependent fashion inhibited the intrinsic phosphatase activity, particularly that of trypsinized calcineurin. SDS in low concentrations (0.005%) augmented the phosphatase activity and inhibited it at high concentrations. Mn2+ (+/- calmodulin) and Ca2+ only with calmodulin present increased the phosphatase activity assayed with low concentrations of SDS. The EDTA dependent inhibition of intrinsic phosphatase was almost abolished in assays containing SDS. Prior exposure of calcineurin to Mn2+ led to a high activity conformation state of calcineurin that was 'long-lived' or 'pseudo-irreversible'. Such Mn2(+)-activated state of calcineurin exhibited no discernible change in the affinity towards myelin basic protein or its inhibition by trifluoperazine. At alkaline pH, Mg2+ supported the intrinsic phosphatase activity, although to a lesser degree than Mn2+. The latter cation, compared to Mg2+ and Ni2+, was also a more powerful stimulator of the calcineurin phosphatase assayed with histone (III-S) and myosin light chain as substrates.

Cloning of human calcineurin A: evidence for two isozymes and identification of a polyproline structural domain.

Two types (I and II) of cDNAs encoding the large (A) subunit of calcineurin, a calmodulin-regulated protein phosphatase, were isolated from human basal ganglia and brainstem mRNA. The complete sequences of the two calcineurin clones are identical except for a 54-base-pair insert in the type I clone and different 3' ends including part of the coding sequence for the C termini of the two proteins. These findings suggest that calcineurin A consists of at least two isozymes that may result from alternative splicing events. The two forms of the enzyme differ in the C terminus, which contains an inhibitory domain rapidly severed by limited proteolysis. With the exception of an 18-amino acid insert, the central parts of the molecules, which harbor the catalytic domains, are identical and show extended similarities with the entire catalytic subunits of protein phosphatases 1 and 2A, defining a distinct family of protein phosphatases. The 40-residue N-terminal fragment, specific for calcineurin, contains a sequence of 11 successive prolines that is also found in bovine brain calcineurin by peptide sequencing. A role in the calmodulin activation of calcineurin is proposed for this novel structural element.

Isolation and Sequence of a cDNA Clone for Human Calcineurin B, the Ca2+-Binding Subunit of the Ca2+/Calmodulin-Stimulated Protein Phosphatase

We have identified and cloned human cDNA for the Ca2+-binding subunit of calcineurin, the brain isozyme of the Ca2+/calmodulin-stimulated protein phosphatase. The 2.5-kb cDNA has an open reading frame of 510 bp, a leader sequence of at least 500 bp, and a 1,277-bp 3'-noncoding sequence. The deduced sequence of the human protein differs from bovine brain calcineurin B by an additional valine at the carboxyl terminus and substitution of Met-11 and Ser-153 by cysteine. A partial clone of the mouse protein corresponding to amino acids 75-150 was also isolated. This portion of the human and mouse protein sequence is identical, with the DNA sequences showing 94% identity. The respective mRNAs in human and mouse are also of similar size. As was observed with protein levels, mRNA abundance in brain is 20-60 times that found in other tissues with the exception of HeLa cells which, like brain, contain abundant calcineurin B mRNA.

Dephosphorylation of Neuromodulin by Calcineurin

Neuromodulin (p57, GAP-43, F1, B-50) is a major neural-specific, calmodulin binding protein found in brain, spinal cord, and retina that is associated with membranes. Phosphorylation of neuromodulin by protein kinase C causes a significant reduction in its affinity for calmodulin (Alexander, K. A., Cimler, B. M., Meirer, K. E., and Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). It has been proposed that neuromodulin may function to bind and concentrate calmodulin at specific sites within neurons and that activation of protein kinase C causes the release of free calmodulin at high concentrations near its target proteins. It was the goal of this study to determine whether bovine brain contains a phosphoprotein phosphatase that will utilize phosphoneuromodulin as a substrate. Phosphatase activity for phosphoneuromodulin was partially purified from a bovine brain extract using DEAE-Sephacel and Sephacryl S-200 gel filtration chromatography. The neuromodulin phosphatase activity was resolved into two peaks by Affi-Gel Blue chromatography. One of these phosphatases, which represented approximately 60% of the total neuromodulin phosphatase activity, was tentatively identified as calcineurin by its requirement for Ca2+ and calmodulin (CaM) and inhibition of its activity by chlorpromazine. Therefore, bovine brain calcineurin was purified to homogeneity and examined for its phosphatase activity against bovine phosphoneuromodulin. Calcineurin rapidly dephosphorylated phosphoneuromodulin in the presence of micromolar Ca2+ and 3 microM CaM. The apparent Km and Vmax for the dephosphorylation of neuromodulin, measured in the presence of micromolar Ca2+ and 2 microM CaM, were 2.5 microM and 70 nmol Pi/mg/min, respectively, compared to a Km and Vmax of 4 microM and 55 nmol Pi/mg/min, respectively, for myosin light chain under the same conditions. Dephosphorylation of neuromodulin by calcineurin was stimulated 50-fold by calmodulin in the presence of micromolar free Ca2+. Half-maximal stimulation was observed at a calmodulin concentration of 0.5 microM. We propose that phosphoneuromodulin may be a physiologically important substrate for calcineurin and that calcineurin and protein kinase C may regulate the levels of free calmodulin available in neurons.

The dephosphorylation of lens α-crystallin a chain

The present communication reports the presence of a phosphoprotein phosphatase activity in bovine lens preparations which dephosphorylates αAp, the phosphorylated form of αA, one of the α-crystallin polypeptides, in a Ca 2+/calmodulin dependent manner. The activity was found in soluble preparations from epithelial cells but it could not be detected in similar preparations from fiber cells. A 60,000 Mr calmodulin binding polypeptide and a 15,000 Mr polypeptide found in the epithelial cell preparations comigrated in SDS-PAGE with the A and B subunits of bovine brain calcineurin (phosphoprotein phosphatase 2B) respectively. The 15,000 Mr was specifically recognized by an anti-bovine brain calcineurin antiserum. Bovine brain calcineurin was as effective in dephosphorylating αAp as the lens preparations. Thus, it is likely that the activity present in the lens is related to this enzyme. The results indicate that the lens specific polypeptide αA may be subject to metabolic control through phosphorylation and dephosphorylation pathways regulated by cAMP and calcium, respectively. Changes in the activities of these pathways appear to occur during differentiation of the lens epithelial cell and may be related to gene regulation during the differentiation process.

The effects of deletions in the central helix of calmodulin on enzyme activation and peptide binding

Using site-directed mutagenesis we have expressed in Escherichia coli three engineered calmodulins (CaM) containing deletions in the solvent-exposed region of the central helix. These are CaM delta 84, Glu-84 removed; CaM delta 83-84, Glu-83 and Glu-84 removed; and CaM delta 81-84, Ser-81 through Glu-84 removed. The abilities of these proteins to activate skeletal muscle myosin light chain kinase, plant NAD kinase, and bovine brain calcineurin activities were determined, as were their abilities to bind a synthetic peptide based on the calmodulin-binding domain of skeletal muscle myosin light chain kinase. Similar results were obtained with all three deletion proteins. Vm values for enzymes activated by the deletion proteins are all within 10-20% of those values obtained with bacterial control calmodulin. Relative to bacterial control values, changes in Kact or Kd values associated with the deletions are all less than an order of magnitude: Kact values for NAD kinase and myosin light chain kinase are increased 5-7-fold, Kd values for binding of the synthetic peptide are increased 4-7-fold, and Kact values for calcineurin are increased only 1-3-fold. In assays of NAD kinase and myosin light chain kinase activation some differences between bovine calmodulin and bacterial control calmodulin were observed. With NAD kinase, Kact values for the bacterial control protein are increased 4-fold relative to values for bovine calmodulin, and Vm values are increased by 50%; with myosin light chain kinase, Kact values are increased 2-fold and Vm values are decreased 10-15% relative to those values obtained with bovine calmodulin. These differences between bacterial control and bovine calmodulins probably can be attributed to known differences in postranslational processing of calmodulin in bacterial and eucaryotic cells. No differences between bovine and control calmodulins were observed in assays of calcineurin activation or peptide binding. Our observations indicate that contacts with the deleted residues, Ser-81 through Glu-84, are not critical in the calmodulin-target complexes we have evaluated. Formation of these calmodulin-target complexes also does not appear to be greatly affected by the global alterations in the structure of calmodulin that are associated with the deletions. In models in which the central helix is maintained in the altered calmodulins, each deleted residue causes the two lobes of calmodulin to be twisted 100 degrees relative to one another and brought 1.5 A closer together.(ABSTRACT TRUNCATED AT 400 WORDS)