Structural basis of calcineurin activation by calmodulin

Calcineurin is the only known calmodulin (CaM) activated protein phosphatase, which is involved in the regulation of numerous cellular and developmental processes and in calcium-dependent signal transduction. Although commonly assumed that CaM displaces the autoinhibitory domain (AID) blocking substrate access to its active site, the structural basis underlying activation remains elusive. We have created a fused ternary complex (CBA) by covalently linking three polypeptides: CaM, calcineurin regulatory B subunit (CnB) and calcineurin catalytic A subunit (CnA). CBA catalytic activity is comparable to that of fully activated native calcineurin in the presence of CaM. The crystal structure showed virtually no structural change in the active site and no evidence of CaM despite being covalently linked. The asymmetric unit contains four molecules; two parallel CBA pairs are packed in an antiparallel mode and the large cavities in crystal packing near the calcineurin active site would easily accommodate multiple positions of AID-bound CaM. Intriguingly, the conformation of the ordered segment of AID is not altered by CaM; thus, it is the disordered part of AID, which resumes a regular α-helical conformation upon binding to CaM, which is displaced by CaM for activation. We propose that the structural basis of calcineurin activation by CaM is through displacement of the disordered fragment of AID which otherwise impedes active site access.     

Structural basis for activation of calcineurin by calmodulin

The highly conserved phosphatase calcineurin (CaN) plays vital roles in numerous processes including T-cell activation, development and function of the central nervous system, and cardiac growth. It is activated by the calcium sensor calmodulin (CaM). CaM binds to a regulatory domain (RD) within CaN, causing a conformational change that displaces an autoinhibitory domain (AID) from the active site, resulting in activation of the phosphatase. This is the same general mechanism by which CaM activates CaM-dependent protein kinases. Previously published data have hinted that the RD of CaN is intrinsically disordered. In this work, we demonstrate that the RD is unstructured and that it folds upon binding CaM, ousting the AID from the catalytic site. The RD is 95 residues long, with the AID attached to its C-terminal end and the 24-residue CaM binding region toward the N-terminal end. This is unlike the CaM-dependent protein kinases that have CaM binding sites and AIDs immediately adjacent in sequence. Our data demonstrate that not only does the CaM binding region folds but also an ～25- to 30-residue region between it and the AID folds, resulting in over half of the RD adopting α-helical structure. This appears to be the first observation of CaM inducing folding of this scale outside of its binding site on a target protein.     

Methionine oxidation in the calmodulin-binding domain of calcineurin disrupts calmodulin binding and calcineurin activation

Calcineurin is a Ca (2+)/calmodulin-activated Ser/Thr phosphatase important in cellular actions resulting in memory formation, cardiac hypertrophy, and T-cell activation. This enzyme is subject to oxidative inactivation by superoxide at low micromolar concentrations and by H 2O 2 at low millimolar concentrations. On the basis of the hypothesis that oxidation of Met residues in calmodulin-binding domains inhibits binding to calmodulin, purified calcineurin was used to study the susceptibility of Met residues to oxidation by H 2O 2. The rate for oxidation of Met 406 in the calmodulin-binding domain was determined to be 4.4 x 10 (-3) M (-1) s (-1), indicating a high susceptibility to oxidation. Functional repercussions of Met 406 oxidation were evaluated using native enzyme and a calcineurin mutant in which Met 406 was exchanged for Leu. Measurement of fluorescent calmodulin binding demonstrated that oxidation of Met 406 results in a 3.3-fold decrease in the affinity of calmodulin for calcineurin. Calcineurin activation exhibited a loss in cooperativity with respect to calmodulin following Met 406 oxidation as shown by a reduction in the Hill slope from 1.88 to 0.86. Maximum phosphatase activity was unaffected by Met oxidation. Changes in the calcineurin-calmodulin interaction were accompanied by a 40% loss in the ability of calmodulin to stimulate binding of immunophilin/immunosuppressant to calcineurin. All effects on calmodulin binding to the native enzyme by the treatment with H 2O 2 could be reversed by treating the enzyme with methionine sulfoxide reductase. These results indicate that the calmodulin-binding domain of calcineurin is susceptible to oxidation at Met 406 and that oxidation disrupts calmodulin binding and enzyme activation. Oxidation-dependent decreases in the affinity of calmodulin for calcineurin can potentially modulate calmodulin-dependent signaling and calmodulin distribution.     

Kinetics of calmodulin binding to calcineurin

Calcineurin (CaN) binds Ca(2+)-saturated calmodulin (CaM) with relatively high affinity; however, an accurate steady-state K(d) value has not been determined. In this report, we describe, using steady-state and stopped-flow fluorescence techniques, the rates of association and dissociation of Ca(2+)-saturated CaM from CaN heterodimer (CaNA/CaNB) and CaNA only. The rate of Ca(2+)/CaM association was determined to be 4.6 x 10(7) M(-1)s(-1). The rate of Ca(2+)/CaM dissociation from CaN was slower than previously reported and was approximately 0.0012 s(-1). In preparations of CaNA alone (no regulatory CaNB subunit), the dissociation rate was slowed further to 0.00026 s(-1). From these data we calculate a K(d) for binding of Ca(2+)-saturated CaM to CaN of 28 pM. This K(d) is significantly lower than previously reported estimates of approximately 1 nM and indicates that CaN is one of the highest affinity CaM-binding proteins identified to date.     

Tau binds both subunits of calcineurin, and binding is impaired by calmodulin

Calcineurin, an important protein Ser/Thr phosphatase which acts on tau in vivo, is a heterodimer of a catalytic subunit, calcineurin A, and a regulatory subunit, calcineurin B, and is unique in being regulated by calmodulin. Here, we find that both subunits of calcineurin bind tau, and calmodulin interferes with the association between calcineurin and tau. The domains of both subunits of calcineurin and tau involved in binding are mapped. We also investigate the functional consequences of the interactions between both subunits of calcineurin, tau and calmodulin, and reveal the interactions affect dephosphorylation of tau by calcineurin and contribute to the balance of phosphorylation and dephosphorylation of tau in vivo. Our findings may be of potential significance in neuronal physiology and also in neurodegenerative disorders. They shed some light on how the interactions might control the phosphorylation state of tau under physiological conditions, and provide new insights into the treatment of tauopathies such as Alzheimer's disease.     

Caerulein-induced intracellular pancreatic zymogen activation is dependent on calcineurin

Aberrant cytosolic Ca(2+) flux in pancreatic acinar cells is critical to the pathological pancreatic zymogen activation observed in acute pancreatitis, but the downstream effectors are not known. In this study, we examined the role of Ca(2+)-activated protein phosphatase 2B (or calcineurin) in zymogen activation. Isolated pancreatic acinar cells were stimulated with supraphysiological caerulein (100 nM) with or without the calcineurin inhibitors FK506 or cell-permeable calcineurin inhibitory peptide (CiP). Chymotrypsin activity was measured as a marker of zymogen activation, and the percent amylase secretion was used as a measure of enzyme secretion. Cytosolic Ca(2+) changes were recorded in acinar cells loaded with the intermediate Ca(2+)-affinity dye fluo-5F using a scanning confocal microscope. A 50% reduction in chymotrypsin activity was observed after pretreatment with 1 microM FK506 or 10 microM CiP. These pretreatments did not affect amylase secretion or the rise in cytosolic Ca(2+) after caerulein stimulation. These findings suggest that calcineurin mediates caerulein-induced intra-acinar zymogen activation but not enzyme secretion or the initial caerulein-induced cytosolic Ca(2+) signal.     

The kinesin-like calmodulin binding protein is differentially involved in cell division

The kinesin-like calmodulin (CaM) binding protein (KCBP), a minus end-directed microtubule motor protein unique to plants, has been implicated in cell division. KCBP is negatively regulated by Ca(2)+ and CaM, and antibodies raised against the CaM binding region inhibit CaM binding to KCBP in vitro; therefore, these antibodies can be used to activate KCBP constitutively. Injection of these antibodies into Tradescantia virginiana stamen hair cells during late prophase induces breakdown of the nuclear envelope within 2 to 10 min and leads the cell into prometaphase. However, mitosis is arrested, and the cell does not progress into anaphase. Injection of antibodies later during cell division has no effect on anaphase transition but causes aberrant phragmoplast formation and delays the completion of cytokinesis by approximately 15 min. These effects are achieved without any apparent degradation of the microtubule cytoskeleton. We propose that during nuclear envelope breakdown and anaphase, activated KCBP promotes the formation of a converging bipolar spindle by sliding and bundling microtubules. During metaphase and telophase, we suggest that its activity is downregulated.     

Phosphorylation of calcineurin: effect on calmodulin binding

The effect of phosphorylation of calcineurin on calmodulin (CaM) binding was examined using a synthetic peptide which contains the CaM-binding domain and the serine phosphorylation site. The peptide, corresponding to residues 391-414 of brain calcineurin A subunit, was rapidly phosphorylated by protein kinase C and Ca2+/CaM-dependent protein kinase II but not by cAMP-dependent protein kinase. Phosphorylation of peptide 391-414 did not significantly alter the binding of CaM when compared to the non-phosphorylated peptide.     

Caspase activation is involved in chronic periodontitis

Periodontitis, a common infectious disease, is initiated by various gram-negative bacteria and characterized by the destruction of the periodontal tissue. Here, we investigated the role of caspases, intracellular proteases that are the key mediators of apoptosis. We show that activation of caspase-3 and caspase-7 is considerably enhanced in gingival tissue from patients with periodontitis. We also demonstrate in in vitro experiments that various periodontopathic bacteria exert a direct growth-suppressing effect and, moreover, can trigger a host-mediated cytotoxic activity involving the CD95 death receptor. Our data suggest that caspase activation is a prominent feature in periodontitis-associated tissue injury.     

The calcineurin inhibitor RCAN1 is involved in cultured macrophage and in vivo immune response

Studies on the role of regulator of calcineurin 1 (RCAN1) in immunity are limited, but have demonstrated an involvement in T-lymphocyte function. Here, we expand these studies to macrophages and in vivo infection. The treatment of RAW and primary mouse macrophages with lipopolysaccharide from Escherichia coli strongly induced RCAN1 isoform 4 (RCAN1-4), but not isoform 1. RCAN1-4 induction involved calcium, calcineurin, and reactive oxygen species. Subsequent analysis with whole bacteria including gram-negative E. coli and gram-positive Staphylococcus aureus revealed strong RCAN1-4 inductions by both, and where tested, dependence on calcium. Staphylococcus aureus cell wall components peptidoglycan and lipoteichoic acid also strongly induced RCAN1-4. In vivo, a significant induction in the proinflammatory cytokines monocyte chemotactic protein-1, interleukin-6, interferon-γ, and tumor necrosis factor-α was observed in knockout (KO) lung vs. wild-type (WT) mice 7 days after nasal infection with Fransicella tularensis. This induction was not accompanied by a significant increase in F. tularensis burden in the KO lung. Additionally, a modest increase in respiratory burst activity in KO vs. WT macrophages was observed. Combined, these studies indicate that RCAN1 is involved in macrophage and the overall in vivo immune response, and provide additional evidence that RCAN1 plays an important role in cell immunity and infectious disease.     

The complex structure of calmodulin bound to a calcineurin peptide

The activity of the protein phosphatase calcineurin (CN) is regulated by an autoinhibition mechanism wherein several domains from its catalytic A subunit, including the calmodulin binding domain (CaMBD), block access to its active site. Upon binding of Ca2+ and calmodulin (Ca2+/CaM) to CaMBD, the autoinhibitory domains dissociate from the catalytic groove, thus activating the enzyme. To date, the structure of the CN/CaM/Ca2+ complex has not been determined in its entirety. Previously, we determined the structure of a fusion protein consisting of CaM and a 25-residue peptide taken from the CaMBD, joined by a 5-glycine linker. This structure revealed a novel CaM binding motif. However, the presence of the extraneous glycine linker cast doubt on the authenticity of this structure as an accurate representation of CN/CaM binding in vivo. Thus, here, we have determined the crystal structure of CaM complexed with the 25-residue CaMBD peptide without the glycine linker at a resolution of 2.1 A. The structure is essentially identical to the fusion construction which displays CaM bound to the CaMBD peptide as a dimer with an open, elongated conformation. The N-lobe from one molecule and C-lobe from another encompass and bind the CaMBD peptide. Thus, it validates the existence of this novel CaM binding motif. Our experiments suggest that the dimeric CaM/CaMBD complex exists in solution, which is unambiguously validated using a carefully-designed CaM-sepharose pull-down experiment. We discuss structural features that produce this novel binding motif, including the role of the CaMBD peptide residues Arg-408, Val-409, and Phe-410, which work to provide rigidity to the otherwise flexible central CaM helix joining the N- and C-lobes, ultimately keeping these lobes apart and forcing "head-to-tail" dimerization to attain the requisite N- and C-lobe pairing for CaMBD binding.     

Survival of mycobacteria in macrophages is mediated by coronin 1-dependent activation of calcineurin

Pathogenic mycobacteria survive within macrophages by avoiding lysosomal delivery, instead residing in mycobacterial phagosomes. Upon infection, the leukocyte-specific protein coronin 1 is actively recruited to mycobacterial phagosomes, where it blocks lysosomal delivery by an unknown mechanism. Analysis of macrophages from coronin 1-deficient mice showed that coronin 1 is dispensable for F-actin-dependent processes such as phagocytosis, motility, and membrane ruffling. However, upon mycobacterial infection, coronin 1 was required for activation of the Ca(2+)-dependent phosphatase calcineurin, thereby blocking lysosomal delivery of mycobacteria. In the absence of coronin 1, calcineurin activity did not occur, resulting in lysosomal delivery and killing of mycobacteria. Furthermore, blocking calcineurin activation with cyclosporin A or FK506 led to lysosomal delivery and intracellular mycobacterial killing. These results demonstrate a role for coronin 1 in activating Ca(2+) dependent signaling processes in macrophages and reveal a function for calcineurin in the regulation of phagosome-lysosome fusion upon mycobacterial infection.     

Alternative splice variant of gamma-calmodulin-dependent protein kinase II alters activation by calmodulin

Calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous, multifunctional enzyme family involved in the regulation of a variety of Ca(2+)-signaling pathways. These family members are expressed from four highly homologous genes (alpha, beta, gamma, and delta) with similar catalytic properties. Additional isoforms of each gene, created by alternative splicing of variable regions I-XI, are differentially expressed in various cell types. gammaB, gammaC, gammaD, gammaE, gammaF, gammaGs, and gammaH CaMKII isoforms are expressed in the biliary epithelium; however, little is known about their roles in these cells. We began our studies into the function of these variable regions by examining the effects of variable region I on kinase activation and calmodulin binding. Activities and calmodulin binding properties of gammaB and gammaGs, which differ only by the exclusion or inclusion of this region, were compared. The K(0.5) for calmodulin was 2.5-fold lower for gammaGs than gammaB. In contrast, gammaB bound calmodulin more tightly in a calmodulin overlay assay. Mutation of variable regions I's charged residue, gammaGs-R318E, resulted in an enzyme with intermediate activation properties but a calmodulin affinity similar to gammaB. Thus, variable region I appears to modulate calmodulin sensitivity, in part, through charge-charge interactions. This altered threshold of activation may modulate cellular responses to gradients of Ca(2+)/calmodulin in the biliary tract.     

Janus kinase 2 is involved in lipopolysaccharide-induced activation of macrophages

The mechanisms by which lipopolysaccharide (LPS) is recognized, and how such recognition leads to innate immune responses, are poorly understood. Stimulation with LPS induces the activation of a variety of proteins, including mitogen-activated protein kinases (MAPKs) and NF-B. Activation of protein tyrosine kinases (PTKs) is also necessary for a number of biological responses to LPS. We used a murine macrophage-like cell line, RAW264.7, to demonstrate that Janus kinase (JAK)2 is tyrosine phosphorylated immediately after LPS stimulation. Anti-Toll-like receptor (TLR)4 neutralization antibody inhibits the phosphorylation of JAK2 and the c-Jun NH₂-terminal protein kinase (JNK). Both the JAK inhibitor AG490 and the kinase-deficient JAK2 protein reduce the phosphorylation of JNK and phosphatidylinositol 3-kinase (PI3K) via LPS stimulation. Pharmacological inhibition of the kinase activity of PI3K with LY-294002 decreases the phosphorylation of JNK. Finally, we show that JAK2 is involved in the production of IL-1 and IL-6. PI3K and JNK are also important for the production of IL-1. These results suggest that LPS induces tyrosine phosphorylation of JAK2 via TLR4 and that JAK2 regulates phosphorylation of JNK mainly through activation of PI3K. Phosphorylation of JAK2 via LPS stimulation is important for the production of IL-1 via the PI3K/JNK cascade. Thus JAK2 plays a pivotal role in LPS-induced signaling in macrophages. cytokine; toll-like receptor-4; c-Jun NH₂-terminal kinase     

Effects of interaction with calcineurin on the reactivities of calmodulin lysines

Calmodulin was trace labeled by acetylation with [3H]acetic anhydride in the presence and absence of a 30% molar excess of the phosphatase calcineurin; phenylalanine was included in the reaction mixtures as an internal standard. The level of 3H acetylation of each of the 7 lysines was determined and corrected for differences arising from reaction conditions using the labeling of the internal standard, following procedures that are closely similar to those used in a previous study of the interaction of calmodulin with myosin light chain kinase (Jackson, A. E., Carraway, K. L., III, Puett, D., and Brew, K. (1986) J. Biol. Chem. 261, 12226-12232). The interaction with calcineurin was found to produce a 10-fold reduction in the acetylation of lysine 75, with lesser but significant effects on lysines 21 and 148. A small but reproducible perturbation of lysine 77 was also observed. The results are similar to those that are produced by the interaction with myosin light chain kinase. However, when they are compared with two recent reports between which there are major discrepancies (Manalan, A. S., and Klee, C. B. (1987) Biochemistry 26, 1382-1390; Winkler, M. A., Fried, V. A., Merat, D. L., and Cheung, W. Y. (1987) J. Biol. Chem. 262, 15466-15471), our results are in good agreement with those obtained in the former study. From the location of the perturbed groups in the three-dimensional structure of calmodulin, it appears that the interaction site on calmodulin for calcineurin, as well as for myosin light chain kinase, is very extended and may include hydrophobic pockets at homologous sites near the carboxyl-terminal ends of the two halves of the molecule.     

Dystroglycan receptor is involved in integrin activation in intestinal epithelia

The dystroglycans (alpha-DG and beta-DG), which play important roles in the formation of basement membranes, have been well studied in skeletal muscle and nerve, but their expression and localization in intestinal epithelial cells has not been previously investigated. Here, we demonstrated that the DG complex, composed of alpha-DG, beta-DG, and utrophin, is specifically expressed in the basolateral membrane of the Caco-2-BBE monolayer. The DG complex coprecipitated with beta(1)-integrin, suggesting a possible interaction among these proteins. In addition, we observed that activation of DG receptors by laminin-1 enhanced the interaction between beta(1)-integrin and laminin-1, whereas activation of DG receptors by laminin-2 reduced the interaction between beta(1)-integrin and laminin-2. Finally, we demonstrated that the intracellular COOH-terminal tail of beta-DG and its binding to the DG binding domain of utrophin are crucial for the interactions between laminin-1/-2 and beta(1)-integrin. Collectively, these novel results indicate that dystroglycans play important roles in the regulation of interactions between intestinal epithelial cells and the extracellular matrix.     

Phosphatidylglycerol is involved in the dimerization of photosystem II

Photosystem II core dimers (450 kDa) and monomers (230 kDa) consisting of CP47, CP43, the D1 and D2 proteins, the extrinsic 33-kDa subunit, and the low molecular weight polypeptides PsbE, PsbF, PsbH, PsbI, PsbK, PsbL, PsbTc, and PsbW were isolated by sucrose density gradient centrifugation. The photosystem II core dimers were treated with phospholipase A2 (PL-A2), which cuts phosphatidylglycerol (PG) and phosphatidylcholine molecules at the sn-2 position. The PL-A2-treated dimers dissociated into two core monomers and further, yielding a CP47-D1-D2 subcomplex and CP43. Thin layer chromatography showed that photosystem II dimers contained four times more PG than their monomeric counterparts but with similar levels of phosphatidylcholine. Consistent with this was the finding that, compared with monomers, the dimers contained a higher level of trans-hexadecanoic fatty acid (C16:1Delta3tr), which is specific to PG of the thylakoid membrane. Moreover, treatment of dimers with PL-A2 increased the free level of this fatty acid specific to PG compared with untreated dimers. Further evidence that PG is involved in stabilizing the dimeric state of photosystem II comes from reconstitution experiments. Using size exclusion chromatography, it was shown that PG containing C16:1Delta3tr, but not other lipid classes, induced significant dimerization of isolated photosystem II monomers. Moreover, this dimerization was observed by electron crystallography when monomers were reconstituted into thylakoid lipids containing PG. The unit cell parameters, p2 symmetry axis, and projection map of the reconstituted dimer was similar to that observed for two-dimensional crystals of the native dimer.