Ca(2+)-dependent and Ca(2+)-independent calmodulin binding sites in erythrocyte protein 4.1. Implications for regulation of protein 4.1 interactions with transmembrane proteins

In vitro protein binding assays identified two distinct calmodulin (CaM) binding sites within the NH(2)-terminal 30-kDa domain of erythrocyte protein 4.1 (4.1R): a Ca(2+)-independent binding site (A(264)KKLWKVCVEHHTFFRL) and a Ca(2+)-dependent binding site (A(181)KKLSMYGVDLHKAKDL). Synthetic peptides corresponding to these sequences bound CaM in vitro; conversely, deletion of these peptides from a 30-kDa construct reduced binding to CaM. Thus, 4.1R is a unique CaM-binding protein in that it has distinct Ca(2+)-dependent and Ca(2+)-independent high affinity CaM binding sites. CaM bound to 4.1R at a stoichiometry of 1:1 both in the presence and absence of Ca(2+), implying that one CaM molecule binds to two distinct sites in the same molecule of 4.1R. Interactions of 4.1R with membrane proteins such as band 3 is regulated by Ca(2+) and CaM. While the intrinsic affinity of the 30-kDa domain for the cytoplasmic tail of erythrocyte membrane band 3 was not altered by elimination of one or both CaM binding sites, the ability of Ca(2+)/CaM to down-regulate 4. 1R-band 3 interaction was abrogated by such deletions. Thus, regulation of protein 4.1 binding to membrane proteins by Ca(2+) and CaM requires binding of CaM to both Ca(2+)-independent and Ca(2+)-dependent sites in protein 4.1.     

Calcium-Dependent Interaction of Calmodulin with Human 80S Ribosomes and Polyribosomes

Ribosomes are the protein factories of every living cell. The process of protein translation is highly complex and tightly regulated by a large number of diverse RNAs and proteins. Earlier studies indicate that Ca(2+) plays a role in protein translation. Calmodulin (CaM), a ubiquitous Ca(2+)-binding protein, regulates a large number of proteins participating in many signaling pathways. Several 40S and 60S ribosomal proteins have been identified to interact with CaM, and here, we report that CaM binds with high affinity to 80S ribosomes and polyribosomes in a Ca(2+)-dependent manner. No binding is observed in buffer with 6 mM Mg(2+) and 1 mM EGTA that chelates Ca(2+), suggesting high specificity of the CaM-ribosome interaction dependent on the Ca(2+) induced conformational change of CaM. The interactions between CaM and ribosomes are inhibited by synthetic peptides comprising putative CaM-binding sites in ribosomal proteins S2 and L14. Using a cell-free in vitro translation system, we further found that these synthetic peptides are potent inhibitors of protein synthesis. Our results identify an involvement of CaM in the translational activity of ribosomes.     

Protein Ser/Thr phosphatases PPEF interact with calmodulin

Regulation of protein dephosphorylation by cytoplasmic Ca(2+) levels and calmodulin (CaM) is well established and considered to be mediated solely by calcineurin. Yet, recent identification of protein phosphatases with EF-hand domains (PPEF/rdgC) point to the existence of another group of Ca(2+)-dependent protein phosphatases. We have recently hypothesised that PPEF/rdgC phosphatases might possess CaM-binding sites of the IQ-type in their N-terminal domains. We now employed yeast two-hybrid system and surface plasmon resonance (SPR) to test this hypothesis. We found that entire human PPEF2 interacts with CaM in the in vivo tests and that its N-terminal domain binds to CaM in a Ca(2+)-dependent manner with nanomolar affinity in vitro. The fragments corresponding to the second exons of PPEF1 and PPEF2, containing the IQ motifs, are sufficient for specific Ca(2+)-dependent interaction with CaM both in vivo and in vitro. These findings demonstrate the existence of mammalian CaM-binding protein Ser/Thr phosphatases distinct from calcineurin and suggest that the activity of PPEF phosphatases may be controlled by Ca(2+) in a dual way: via C-terminal Ca(2+)-binding domain and via interaction of the N-terminal domain with CaM.     

Binding and activation of nitric oxide synthase isozymes by calmodulin EF hand pairs

Calmodulin (CaM) is a cytosolic Ca(2+) signal-transducing protein that binds and activates many different cellular enzymes with physiological relevance, including the nitric oxide synthase (NOS) isozymes. CaM consists of two globular domains joined by a central linker; each domain contains an EF hand pair. Four different mutant CaM proteins were used to investigate the role of the two CaM EF hand pairs in the binding and activation of the mammalian inducible NOS (iNOS) and the constitutive NOS (cNOS) enzymes, endothelial NOS (eNOS) and neuronal NOS (nNOS). The role of the CaM EF hand pairs in different aspects of NOS enzymatic function was monitored using three assays that monitor electron transfer within a NOS homodimer. Gel filtration studies were used to determine the effect of Ca(2+) on the dimerization of iNOS when coexpressed with CaM and the mutant CaM proteins. Gel mobility shift assays were performed to determine binding stoichiometries of CaM proteins to synthetic NOS CaM-binding domain peptides. Our results show that the N-terminal EF hand pair of CaM contains important binding and activating elements for iNOS, whereas the N-terminal EF hand pair in conjunction with the central linker region is required for cNOS enzyme binding and activation. The iNOS enzyme must be coexpressed with wild-type CaM in vitro because of its propensity to aggregate when residues of the highly hydrophobic CaM-binding domain are exposed to an aqueous environment. A possible role for iNOS aggregation in vivo is also discussed.     

Microtubule sliding movement in tilapia sperm flagella axoneme is regulated by Ca2+/calmodulin-dependent protein phosphorylation

Demembranated euryhaline tilapia Oreochromis mossambicus sperm were reactivated in the presence of concentrations in excess of 10(-6) M Ca(2+). Motility features changed when Ca(2+) concentrations were increased from 10(-6) to 10(-5) M. Although the beat frequency did not increase, the shear angle and wave amplitude of flagellar beating increased, suggesting that the sliding velocity of microtubules in the axoneme, which represents dynein activity, rises with an increase in Ca(2+). Thus, it is possible that Ca(2+) binds to flagellar proteins to activate flagellar motility as a result of the enhanced dynein activity. One Ca(2+)-binding protein (18 kDa, pI 4.0), calmodulin (CaM), was detected by (45)Ca overlay assay and immunologically. A CaM antagonist, W-7, suppressed the reactivation ratio and swimming speed, suggesting that the 18 kDa Ca(2+)-binding protein is CaM and that CaM regulates flagellar motility. CaMKIV was detected immunologically as a single 48 kDa band in both the fraction of low ion extract of the axoneme and the remnant of the axoneme, suggesting that CaMKIV binds to distinct positions in the axoneme. It is possible that CaMKIV phosphorylates the axonemal proteins in a Ca(2+)/CaM-dependent manner for regulating the dynein activity. A (32)P-uptake in the axoneme showed that 48, 75, 120, 200, 250, 380, and 400 kDa proteins were phosphorylated in a Ca(2+)/CaM kinase-dependent manner. Proteins (380 kDa) were phosphorylated in the presence of 10(-5) M Ca(2+). It is possible that an increase in Ca(2+) induces Ca(2+)/CaM kinase-dependent regulation, including protein phosphorylation for activation/regulation of dynein activity in flagellar axoneme.     

Calmodulin is involved in the Ca2+-dependent activation of ceramide kinase as a calcium sensor

We recently demonstrated that the activation of ceramide kinase (CERK) and the formation of its product, ceramide 1-phosphate (C1P), are necessary for the degranulation pathway in mast cells and that the kinase activity of this enzyme is completely dependent on the intracellular concentration of Ca(2+) (Mitsutake, S., Kim, T.-J., Inagaki, Y., Kato, M., Yamashita, T., and Igarashi, Y. (2004) J. Biol. Chem. 279, 17570-17577). Despite the demonstrated importance of Ca(2+) as a regulator of CERK activity, there are no apparent binding domains in the enzyme and the regulatory mechanism has not been well understood. In the present study, we found that calmodulin (CaM) is involved in the Ca(2+)-dependent activation of CERK. The CaM antagonist W-7 decreased both CERK activity and intracellular C1P formation. Additionally, exogenously added CaM enhanced CERK activity even at low concentrations of Ca(2+). The CERK protein was co-immunoprecipitated with an anti-CaM antibody, indicating formation of intracellular CaM.CERK complexes. An in vitro CaM binding assay also demonstrated Ca(2+)-dependent binding of CaM to CERK. These results strongly suggest that CaM acts as a Ca(2+) sensor for CERK. Furthermore, a CaM binding assay using various mutants of CERK revealed that the binding site of CERK is located within amino acids 422-435. This region appears to include a type 1-8-14B CaM binding motif and is predicted to form an amphipathic helical wheel, which is utilized in CaM recognition. The expression of a deletion mutant of CERK that contained the CaM binding domain but lost CERK activity inhibited the Ca(2+)-dependent C1P formation. These results suggest that this domain could saturate the CaM and hence block Ca(2+)-dependent activation of CERK. Finally, we reveal that in mast cell degranulation CERK acts downstream of CaM, similar to CaM-dependent protein kinase II, which had been assumed to be the main target of CaM in mast cells.     

Ca(2+)-calmodulin-dependent protein kinases Ia and Ib from rat brain I. Identification, purification, and structural comparisons.

Two Ca(2+)-calmodulin (CaM)-dependent protein kinases were purified from rat brain using as substrate a synthetic peptide based on site 1 (site 1 peptide) of the synaptic vesicle-associated protein, synapsin I. One of the purified enzymes was an approximately 89% pure protein of M(r) = 43,000 which bound CaM in a Ca(2+)-dependent fashion. The other purified enzyme was an apparently homogenous protein of M(r) = 39,000 accompanied by a small amount of a M(r) = 37,000 form which may represent a proteolytic product of the 39-kDa enzyme. The 39-kDa protein bound CaM in a Ca(2+)-dependent fashion. Gel filtration analysis indicated that both enzymes are monomers. The 43- and 39-kDa enzymes are named Ca(2+)-CaM-dependent protein kinases Ia and Ib (CaM kinases Ia, Ib), respectively. The specific activities of CaM kinases Ia and Ib were similar (5-8 mumol/min/mg protein). CaM kinase Ia (but not CaM kinase Ib) activity was enhanced by addition of a CaM-Sepharose column wash (non-binding) fraction suggesting the existence of an "activator" of CaM kinase Ia. Both kinases phosphorylated exogenous substrates (site 1 peptide and synapsin I) in a Ca(2+)-CaM-dependent fashion and both kinases underwent autophosphorylation. CaM kinase Ia autophosphorylation was Ca(2+)-CaM-dependent and occurred exclusively on threonine while CaM kinase Ib autophosphorylation showed Ca(2+)-CaM independence and occurred on both serine and threonine. Proteolytic digestion of autophosphorylated CaM kinases Ia and Ib yielded phosphopeptides of differing M(r). These characteristics, as well as enzymatic and regulatory properties (DeRemer, M. F., Saeli, R. J. Brautigen, D. L., and Edelman, A. M. (1992) J. Biol. Chem. 267, 13466-13471), indicate that CaM kinases Ia and Ib are distinct and possibly previously unrecognized enzymes.     

Mlo, a modulator of plant defense and cell death, is a novel calmodulin-binding protein. Isolation and characterization of a rice Mlo homologue

Transient influx of Ca(2+) constitutes an early event in the signaling cascades that trigger plant defense responses. However, the downstream components of defense-associated Ca(2+) signaling are largely unknown. Because Ca(2+) signals are mediated by Ca(2+)-binding proteins, including calmodulin (CaM), identification and characterization of CaM-binding proteins elicited by pathogens should provide insights into the mechanism by which Ca(2+) regulates defense responses. In this study, we isolated a gene encoding rice Mlo (Oryza sativa Mlo; OsMlo) using a protein-protein interaction-based screening of a cDNA expression library constructed from pathogen-elicited rice suspension cells. OsMlo has a molecular mass of 62 kDa and shares 65% sequence identity and scaffold topology with barley Mlo, a heptahelical transmembrane protein known to function as a negative regulator of broad spectrum disease resistance and leaf cell death. By using gel overlay assays, we showed that OsMlo produced in Escherichia coli binds to soybean CaM isoform-1 (SCaM-1) in a Ca(2+)-dependent manner. We located a 20-amino acid CaM-binding domain (CaMBD) in the OsMlo C-terminal cytoplasmic tail that is necessary and sufficient for Ca(2+)-dependent CaM complex formation. Specific binding of the conserved CaMBD to CaM was corroborated by site-directed mutagenesis, a gel mobility shift assay, and a competition assay with a Ca(2+)/CaM-dependent enzyme. Expression of OsMlo was strongly induced by a fungal pathogen and by plant defense signaling molecules. We propose that binding of Ca(2+)-loaded CaM to the C-terminal tail may be a common feature of Mlo proteins.     

Ca2+-free calmodulin and calmodulin damaged by in vitro aging are selectively degraded by 26 S proteasomes without ubiquitination

The ubiquitin-proteasome pathway is believed to selectively degrade post-synthetically damaged proteins in eukaryotic cells. To study this process we used calmodulin (CaM) as a substrate because of its importance in cell regulation and because it acquires isoaspartyl residues in its Ca(2+)-binding regions both in vivo and after in vitro "aging" (incubation for 2 weeks without Ca(2+)). When microinjected into Xenopus oocytes, in vitro aged CaM was degraded much faster than native CaM by a proteasome-dependent process. Similarly, in HeLa cell extracts aged CaM was degraded at a higher rate, even though it was not conjugated to ubiquitin more rapidly than the native species. Ca(2+) stimulated the ubiquitination of both species, but inhibited their degradation. Thus, for CaM, ubiquitination and proteolysis appear to be dissociated. Accordingly, purified muscle 26 S proteasomes could degrade aged CaM and native Ca(2+)-free (apo) CaM without ubiquitination. Addition of Ca(2+) dramatically reduced degradation of the native molecules but only slightly reduced the breakdown of the aged species. Thus, upon Ca(2+) binding, native CaM assumes a non-degradable conformation, which most of the age-damaged species cannot assume. Thus, flexible conformations, as may arise from age-induced damage or the absence of ligands, can promote degradation directly by the proteasome without ubiquitination.     

Structural basis for endothelial nitric oxide synthase binding to calmodulin

The enzyme nitric oxide synthase (NOS) is exquisitely regulated in vivo by the Ca(2+) sensor protein calmodulin (CaM) to control production of NO, a key signaling molecule and cytotoxin. The differential activation of NOS isozymes by CaM has remained enigmatic, despite extensive research. Here, the crystallographic structure of Ca(2+)-loaded CaM bound to a 20 residue peptide comprising the endothelial NOS (eNOS) CaM-binding region establishes their individual conformations and intermolecular interactions, and suggests the basis for isozyme-specific differences. The alpha-helical eNOS peptide binds in an antiparallel orientation to CaM through extensive hydrophobic interactions. Unique NOS interactions occur with: (i). the CaM flexible central linker, explaining its importance in NOS activation; and (ii). the CaM C-terminus, explaining the NOS-specific requirement for a bulky, hydrophobic residue at position 144. This binding mode expands mechanisms for CaM-mediated activation, explains eNOS deactivation by Thr495 phosphorylation, and implicates specific hydrophobic residues in the Ca(2+) independence of inducible NOS.     

Ca2+ signaling regulates ecmB expression, cell differentiation and slug regeneration in Dictyostelium

Ca(2+) regulates cell differentiation and morphogenesis in a diversity of organisms and dysregulation of Ca(2+) signal transduction pathways leads to many cellular pathologies. In Dictyostelium Ca(2+) induces ecmB expression and stalk cell differentiation in vitro. Here we have analyzed the pattern of ecmB expression in intact and bisected slugs and the effect of agents that affect Ca(2+) levels or antagonize calmodulin (CaM) on this expression pattern. We have shown that Ca(2+) and CaM regulate ecmB expression and pstAB/pstB cell differentiation in vivo. Agents that increase intracellular Ca(2+) levels increased ecmB expression and/or pstAB and pstB cell differentiation, while agents that decrease intracellular Ca(2+) or antagonize CaM decreased it. In isolated slug tips agents that affect Ca(2+) levels and antagonize CaM had differential effect on ecmB expression and cell differentiation in the anterior versus posterior zones. Agents that increase intracellular Ca(2+) levels increased the number of ecmB expressing cells in the anterior region of slugs, while agents that decrease intracellular Ca(2+) levels or antagonize CaM activity increased the number of ecmB expressing cells in the posterior. We have also demonstrated that agents that affect Ca(2+) levels or antagonize CaM affect cells motility and regeneration of shape in isolated slug tips and backs and regeneration of tips in isolated slug backs. To our knowledge, this is the first study detailing the pattern of ecmB expression in regenerating slugs as well as the role of Ca(2+) and CaM in the regeneration process and ecmB expression.     

Sequence reversed peptide from CaMKK binds to calmodulin in reversible Ca2+ -dependent manner

Calmodulin (CaM) is a highly versatile Ca(2+) signaling transducer known to regulate over a hundred proteins. In this paper, we further demonstrate the versatility of CaM binding by showing that it binds to a synthetic peptide (revCKKp) made by reversing the amino acid sequence of the CaM-binding peptide (CKKp) from CaM-dependent protein kinase kinase (CaMKK) (residues 438-463). Sequence comparison between revCKKp and other CaM-binding peptides (CBPs) from the CaM target databank showed that revCKKp does not resemble any existing classes of CBPs, except CKKp [M. Zhang, T. Yuan, Molecular mechanisms of calmodulin's functional versatility, Biochem. Cell Biol. 76 (1998) 313-323; S.W. Vetter, E. Leclerc, Novel aspects of calmodulin target recognition and activation, Eur. J. Biochem. 270 (2003) 404-414]. Furthermore, computational modeling showed that revCKKp could bind CaM in a similar manner to CKKp. Lastly, we experimentally showed that our synthetic revCKKp binds to CaM in a reversible Ca(2+)-dependent manner.     

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.     

Ca(2+)/calmodulin directly interacts with the pleckstrin homology domain of AKT1

AKT kinase, also known as protein kinase B, is a key regulator of cell growth, proliferation, and metabolism. The activation of the AKT signaling pathway is one of the most frequent molecular alterations in a wide variety of human cancers. Dickson and coworkers recently observed that Ca(2+).calmodulin (Ca(2+).CaM) may be a common regulator of AKT1 activation (Deb, T. B., Coticchia, C. M., and Dickson, R. B. (2004) J. Biol. Chem. 279, 38903-38911). In our efforts to scan the mRNA-displayed proteome libraries for Ca(2+).CaM-binding proteins, we found that both human and Caenorhabditis elegans AKT1 kinases bound to CaM in a Ca(2+)-dependent manner (Shen, X., Valencia, C. A., Szostak, J., Dong, B., and Liu, R. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 5969-5974 and Shen, X., Valencia, C. A., Gao, W., Cotten, S. W., Dong, B., Chen, M., and Liu, R. (2007) submitted for publication). Here we demonstrate that Ca(2+).CaM and human AKT1 were efficiently co-immunoprecipitated, and their interaction was direct rather than mediated by other proteins. The binding is in part attributed to the first 42 residues of the pleckstrin homology (PH) domain, a region that is critical for the recognition of its lipid ligands. The PH domain of human AKT1 can disrupt the complex of the full-length AKT1 with Ca(2+).CaM. In addition, Ca(2+).CaM competes with phosphatidylinositol 3,4,5-trisphophate for interaction with the PH domain of human AKT1. Our findings suggest that Ca(2+).CaM is directly involved in regulating the functions of AKT1, presumably by releasing the activated AKT1 from the plasma membrane and/or prohibiting it from re-association with phosphoinositides on plasma membrane.     

Calmodulin-dependent and -independent activation of endothelial nitric-oxide synthase by heat shock protein 90

Endothelial nitric oxide synthase (eNOS), which generates the endogenous vasodilator, nitric oxide (NO), is highly regulated by post-translational modifications and protein interactions. Heat shock protein 90 (HSP90) binds directly to eNOS, augmenting NO production. We have used purified proteins to characterize further the mechanism by which HSP90 increases eNOS activity at low (100 nm) and high (10 microm) Ca(2+) levels. In the presence of calmodulin (CaM), HSP90 increased eNOS activity dose dependently at both low and high Ca(2+) concentrations. This effect was abolished by the specific HSP90 inhibitor geldanamycin (GA) at both calcium concentrations. The EC(50) values of eNOS for both Ca(2+) and CaM were decreased in the presence of HSP90. HSP90 also significantly increased the rate of NADPH-dependent cytochrome c reduction by eNOS at both low and high Ca(2+) concentrations. HSP90 bound to eNOS in a dose-dependent manner, and the amount of bound HSP90 also increased with increasing Ca(2+)/CaM. At 100 nm Ca(2+), HSP90 promoted dose-dependent CaM binding to eNOS that was fully inhibitable by GA. At high calcium, HSP90 did not affect CaM binding to eNOS, but GA inhibited HSP90 binding to eNOS. At high Ca(2+), HSP90 caused the V(max) of eNOS for l-arginine to increase by 2-fold, but the K(m) of eNOS was unchanged. HSP90 bound preferentially to CaM-prebound eNOS and significantly increased both its NO synthesis and reductase activities. These data support that HSP90 promotes eNOS activity by two mechanisms: (i) a CaM-dependent mechanism operative at low Ca(2+) concentrations, characterized by an increase in the affinity of eNOS for CaM and (ii) a CaM-independent mechanism apparent at high Ca(2+) concentrations, characterized by stimulation of eNOS reductase activity without further change in CaM binding. These studies contribute to our understanding of eNOS activation by HSP90 and provide a basis for in vitro studies of other eNOS-interacting proteins.     

RC3/Neurogranin and Ca2+/calmodulin-dependent protein kinase II produce opposing effects on the affinity of calmodulin for calcium

The interaction of calmodulin with its target proteins is known to affect the kinetics and affinity of Ca(2+) binding to calmodulin. Based on thermodynamic principles, proteins that bind to Ca(2+)-calmodulin should increase the affinity of calmodulin for Ca(2+), while proteins that bind to apo-calmodulin should decrease its affinity for Ca(2+). We quantified the effects on Ca(2+)-calmodulin interaction of two neuronal calmodulin targets: RC3, which binds both Ca(2+)- and apo-calmodulin, and alphaCaM kinase II, which binds selectively to Ca(2+)-calmodulin. RC3 was found to decrease the affinity of calmodulin for Ca(2+), whereas CaM kinase II increases the calmodulin affinity for Ca(2+). Specifically, RC3 increases the rate of Ca(2+) dissociation from the C-terminal sites of calmodulin up to 60-fold while having little effect on the rate of Ca(2+) association. Conversely, CaM kinase II decreases the rates of dissociation of Ca(2+) from both lobes of calmodulin and autophosphorylation of CaM kinase II at Thr(286) induces a further decrease in the rates of Ca(2+) dissociation. RC3 dampens the effects of CaM kinase II on Ca(2+) dissociation by increasing the rate of dissociation from the C-terminal lobe of calmodulin when in the presence of CaM kinase II. This effect is not seen with phosphorylated CaM kinase II. The results are interpreted according to a kinetic scheme in which there are competing pathways for dissociation of the Ca(2+)-calmodulin target complex. This work indicates that the Ca(2+) binding properties of calmodulin are highly regulated and reveals a role for RC3 in accelerating the dissociation of Ca(2+)-calmodulin target complexes at the end of a Ca(2+) signal.