Matrin 3 is a Ca2+/calmodulin-binding protein cleaved by caspases

Matrin 3 is a nuclear matrix protein that has been implicated in interacting with other nuclear proteins to anchor hyperedited RNAs to the nuclear matrix, in modulating the activity of proximal promoters, and as the main PKA substrate following NMDA receptor activation. In our proteome-wide selections for calmodulin (CaM) binding proteins and for caspase substrates using mRNA-displayed human proteome libraries, matrin 3 was identified as both a Ca(2+)-dependent CaM-binding protein and a downstream substrate of caspases. We report here, the in vitro characterization of the CaM-binding motif and the caspase cleavage site on matrin 3. Significantly, the Ca(2+)/CaM-binding motif is partially overlapped by the RRM of matrin 3 and is also very close to the bipartite NLS that is essential for its nuclear localization. The caspase cleavage site is downstream of the NLS but upstream of the second U1-like zinc finger. Our results suggest that the functions of matrin 3 could be regulated by both Ca(2+)-dependent interaction with CaM and caspase-mediated cleavage.     

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.     

At-ACA8 encodes a plasma membrane-localized calcium-ATPase of Arabidopsis with a calmodulin-binding domain at the N terminus

A Ca(2+)-ATPase was purified from plasma membranes (PM) isolated from Arabidopsis cultured cells by calmodulin (CaM)-affinity chromatography. Three tryptic fragments from the protein were microsequenced and the corresponding cDNA was amplified by polymerase chain reaction using primers designed from the microsequences of the tryptic fragments. At-ACA8 (Arabidopsis-autoinhibited Ca(2+)-ATPase, isoform 8, accession no. AJ249352) encodes a 1,074 amino acid protein with 10 putative transmembrane domains, which contains all of the characteristic motifs of Ca(2+)-transporting P-type Ca(2+)-ATPases. The identity of At-ACA8p as the PM Ca(2+)-ATPase was confirmed by immunodetection with an antiserum raised against a sequence (valine-17 through threonine-31) that is not found in other plant CaM-stimulated Ca(2+)-ATPases. Confocal fluorescence microscopy of protoplasts immunodecorated with the same antiserum confirmed the PM localization of At-ACA8. At-ACA8 is the first plant PM localized Ca(2+)-ATPase to be cloned and is clearly distinct from animal PM Ca(2+)-ATPases due to the localization of its CaM-binding domain. CaM overlay assays localized the CaM-binding domain of At-ACA8p to a region of the N terminus of the enzyme around tryptophan-47, in contrast to a C-terminal localization for its animal counterparts. Comparison between the sequence of At-ACA8p and those of endomembrane-localized type IIB Ca(2+)-ATPases of plants suggests that At-ACA8 is a representative of a new subfamily of plant type IIB Ca(2+)-ATPases.     

Conformation of the calmodulin-binding domain of metabotropic glutamate receptor subtype 7 and its interaction with calmodulin

Calmodulin (CaM), a Ca(2+)-binding protein, is a well-known regulator of various cellular functions. One of the targets of CaM is metabotropic glutamate receptor 7 (mGluR7), which serves as a low-pass filter for glutamate in the pre-synaptic terminal to regulate neurotransmission. Surface plasmon resonance (SPR), circular dichroism (CD) spectroscopy and nuclear magnetic spectroscopy (NMR) were performed to study the structure of the peptides corresponding to the CaM-binding domain of mGluR7 and their interaction with CaM. Unlike well-known CaM-binding peptides, mGluR7 has a random coil structure even in the presence of trifluoroethanol. Moreover, NMR data suggested that the complex between Ca(2+)/CaM and the mGluR7 peptide has multiple conformations. The mGluR7 peptide has been found to interact with CaM even in the absence of Ca(2+), and the binding is directed toward the C-domain of apo-CaM rather than the N-domain. We propose a possible mechanism for the activation of mGluR7 by CaM. A pre-binding occurs between apo-CaM and mGluR7 in the resting state of cells. Then, the Ca(2+)/CaM-mGluR7 complex is formed once Ca(2+) influx occurs. The weak interaction at lower Ca(2+) concentrations is likely to bind CaM to mGluR7 for the fast complex formation in response to the elevation of Ca(2+) concentration.     

A Ca2+/calmodulin-binding peroxidase from Euphorbia latex: novel aspects of calcium-hydrogen peroxide cross-talk in the regulation of plant defenses

Calmodulin (CaM) is a ubiquitous Ca(2+) sensor found in all eukaryotes, where it participates in the regulation of diverse calcium-dependent physiological processes. In response to fluctuations of the intracellular concentration of Ca(2+), CaM binds to a set of unrelated target proteins and modulates their activity. In plants, a growing number of CaM-binding proteins have been identified that apparently do not have a counterpart in animals. Some of these plant-specific Ca(2+)/CaM-activated proteins are known to tune the interaction between calcium and H(2)O(2) in orchestrating plant defenses against biotic and abiotic stresses. We previously characterized a calcium-dependent peroxidase isolated from the latex of the Mediterranean shrub Euphorbia characias (ELP) [Medda et al. (2003) Biochemistry 42, 8909-8918]. Here we report the cDNA nucleotide sequence of Euphorbia latex peroxidase, showing that the derived protein has two distinct amino acid sequences recognized as CaM-binding sites. The cDNA encoding for an E. characias CaM was also found and sequenced, and its protein product was detected in the latex. Results obtained from different CaM-binding assays and the determination of steady-state parameters showed unequivocally that ELP is a CaM-binding protein activated by the Ca(2+)/CaM system. To the best of our knowledge, this is the first example of a peroxidase regulated by this classic signal transduction mechanism. These findings suggest that peroxidase might be another node in the Ca(2+)/H(2)O(2)-mediated plant defense system, having both positive and negative effects in regulating H(2)O(2) homeostasis.     

FRET conformational analysis of calmodulin binding to nitric oxide synthase peptides and enzymes

Calmodulin (CaM) is a ubiquitous Ca (2+)-sensor protein that binds and activates the nitric oxide synthase (NOS) enzymes. We have used fluorescence resonance energy transfer (FRET) to examine the conformational transitions of CaM induced by its binding to synthetic nitric oxide synthase (NOS) CaM-binding domain peptides and full length heme-free constitutive NOS (cNOS) enzymes over a range of physiologically relevant free Ca (2+) concentrations. We demonstrate for the first time that the domains of CaM collapse when associated with Ca (2+)-independent inducible NOS CaM-binding domain, similar to the previously solved crystal structures of CaM bound to the Ca (2+)-dependent cNOS peptides. We show that the association of CaM is not detectable with the cNOS peptides at low free Ca (2+) concentrations (<40 nM). In contrast, we demonstrate that CaM associates with the cNOS holo-enzymes in the absence of Ca (2+) and that the Ca (2+)-dependent transition occurs at a lower free Ca (2+) concentration with the cNOS holo-enzymes. Our results suggest that other regions outside of the CaM-binding domain in the cNOS enzymes are involved in the recruitment and binding of CaM. We also demonstrate that CaM binds to the cNOS enzymes in a sequential manner with the Ca (2+)-replete C-lobe binding first followed by the Ca (2+)-replete N-lobe. This novel FRET study helps to clarify some of the observed similarities and differences between the Ca (2+)-dependent/independent interaction between CaM and the NOS isozymes.     

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.     

Molecular basis of calmodulin tethering and Ca2+-dependent inactivation of L-type Ca2+ channels.

Ca(2+)-dependent inactivation (CDI) of L-type Ca(2+) channels plays a critical role in controlling Ca(2+) entry and downstream signal transduction in excitable cells. Ca(2+)-insensitive forms of calmodulin (CaM) act as dominant negatives to prevent CDI, suggesting that CaM acts as a resident Ca(2+) sensor. However, it is not known how the Ca(2+) sensor is constitutively tethered. We have found that the tethering of Ca(2+)-insensitive CaM was localized to the C-terminal tail of alpha(1C), close to the CDI effector motif, and that it depended on nanomolar Ca(2+) concentrations, likely attained in quiescent cells. Two stretches of amino acids were found to support the tethering and to contain putative CaM-binding sequences close to or overlapping residues previously shown to affect CDI and Ca(2+)-independent inactivation. Synthetic peptides containing these sequences displayed differences in CaM-binding properties, both in affinity and Ca(2+) dependence, leading us to propose a novel mechanism for CDI. In contrast to a traditional disinhibitory scenario, we suggest that apoCaM is tethered at two sites and signals actively to slow inactivation. When the C-terminal lobe of CaM binds to the nearby CaM effector sequence (IQ motif), the braking effect is relieved, and CDI is accelerated.     

Identification of the calmodulin binding domain of connexin 43

Calmodulin (CaM) has been implicated in mediating the Ca(2+)-dependent regulation of gap junctions. This report identifies a CaM-binding motif comprising residues 136-158 in the intracellular loop of Cx43. A 23-mer peptide encompassing this CaM-binding motif was shown to bind Ca(2+)-CaM with 1:1 stoichiometry by using various biophysical approaches, including surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and NMR. Far UV circular dichroism studies indicated that the Cx43-derived peptide increased its alpha-helical contents on CaM binding. Fluorescence and NMR studies revealed conformational changes of both the peptide and CaM following formation of the CaM-peptide complex. The apparent dissociation constant of the peptide binding to CaM in physiologic K(+) is in the range of 0.7-1 microM. Upon binding of the peptide to CaM, the apparent K(d) of Ca(2+) for CaM decreased from 2.9 +/- 0.1 to 1.6 +/- 0.1 microM, and the Hill coefficient n(H) increased from 2.1 +/- 0.1 to 3.3 +/- 0.5. Transient expression in HeLa cells of two different mutant Cx43-EYFP constructs without the putative Cx43 CaM-binding site eliminated the Ca(2+)-dependent inhibition of Cx43 gap junction permeability, confirming that residues 136-158 in the intracellular loop of Cx43 contain the CaM-binding site that mediates the Ca(2+)-dependent regulation of Cx43 gap junctions. Our results provide the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx43 in a Ca(2+)-dependent manner, providing a molecular basis for the well characterized Ca(2+)-dependent inhibition of Cx43-containing gap junctions.     

Arabidopsis ubiquitin-specific protease 6 (AtUBP6) interacts with calmodulin

Calmodulin (CaM), a key Ca(2+) sensor in eukaryotes, regulates diverse cellular processes by interacting with many proteins. To identify Ca(2+)/CaM-mediated signaling components, we screened an Arabidopsis expression library with horseradish peroxidase-conjugated Arabidopsis calmodulin2 (AtCaM2) and isolated a homolog of the UBP6 deubiquitinating enzyme family (AtUBP6) containing a Ca(2+)-dependent CaM-binding domain (CaMBD). The CaM-binding activity of the AtUBP6 CaMBD was confirmed by CaM mobility shift assay, phosphodiesterase competition assay and site-directed mutagenesis. Furthermore, expression of AtUBP6 restored canavanine resistance to the Deltaubp6 yeast mutant. This is the first demonstration that Ca(2+) signaling via CaM is involved in ubiquitin-mediated protein degradation and/or stabilization in plants.     

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.     

Molecular and biochemical analysis of calmodulin interactions with the calmodulin-binding domain of plant glutamate decarboxylase.

We previously provided what to our knowledge is the first evidence that plant glutamate decarboxylase (GAD) is a calmodulin (CaM)-binding protein. Here, we studied the GAD CaM-binding domain in detail. A synthetic peptide of 26 amino acids corresponding to this domain forms a stable complex with Ca2+/CaM with a 1:1 stoichiometry, and amino acid substitutions suggest that tryptophan-485 has an indispensable role in CaM binding. Chemical cross-linking revealed specific CaM/GAD interactions even in the absence of Ca2+. However, increasing KCI concentrations or deletion of two carboxy-terminal lysines abolished these interactions but had a mild effect on CaM/GAD interactions in the presence of Ca2+. We conclude that in the presence of Ca(2+)-hydrophobic interactions involving tryptophan-485 and electrostatic interactions involving the carboxy-terminal lysines mediate CaM/GAD complex formation. By contrast, in the absence of Ca2+, CaM/GAD interactions are essentially electrostatic and involve the carboxy-terminal lysines. In addition, a tryptophan residue and carboxy-terminal lysines are present in the CaM-binding domain of an Arabidopsis GAD. Finally, we demonstrate that petunia GAD activity is stimulated in vitro by Ca2+/CaM. Our study provides a molecular basis for Ca(2+)-dependent CaM/GAD interactions and suggests the possible occurrence of Ca(2+)-independent CaM/GAD interactions.     

Regulation of MAPK phosphatase 1 (AtMKP1) by calmodulin in Arabidopsis

The mitogen-activated protein kinases (MAPKs) are key signal transduction molecules, which respond to various external stimuli. The MAPK phosphatases (MKPs) are known to be negative regulators of MAPKs in eukaryotes. We screened an Arabidopsis cDNA library using horseradish peroxidase-conjugated calmodulin (CaM), and isolated AtMKP1 as a CaM-binding protein. Recently, tobacco NtMKP1 and rice OsMKP1, two orthologs of Arabidopsis AtMKP1, were reported to bind CaM via a single putative CaM binding domain (CaMBD). However, little is known about the regulation of phosphatase activity of plant MKP1s by CaM binding. In this study, we identified two Ca(2+)-dependent CaMBDs within AtMKP1. Specific binding of CaM to two different CaMBDs was verified using a gel mobility shift assay, a competition assay with a Ca(2+)/CaM-dependent enzyme, and a split-ubiquitin assay. The peptides for two CaMBDs, CaMBDI and CaMBDII, bound CaM in a Ca(2+)-dependent manner, and the binding affinity of CaMBDII was found to be higher than that of CaMBDI. CaM overlay assays using mutated CaMBDs showed that four amino acids, Trp(453) and Leu(456) in CaMBDI and Trp(678) and Ile(684) in CaMBDII, play a pivotal role in CaM binding. Moreover, the phosphatase activity of AtMKP1 was increased by CaM in a Ca(2+)-dependent manner. Our results suggest that two important signaling pathways, Ca(2+) signaling and the MAPK signaling cascade, are connected in plants via the regulation of AtMKP1 activity. To our knowledge, this is the first report to show that the biochemical activity of MKP1 in plants is regulated by CaM.     

The calmodulin-binding domain from a plant kinesin functions as a modular domain in conferring Ca2+-calmodulin regulation to animal plus- and minus-end kinesins

Plant kinesin-like calmodulin-binding protein (KCBP) is a novel member of the kinesin superfamily that interacts with calmodulin (CaM) via its CaM-binding domain (CBD). Activated CaM (Ca(2+)-CaM) has been shown to inhibit KCBP interaction with microtubules (MTs) thereby abolishing its motor- and MT-dependent ATPase activities. To test whether the fusion of CBD to non-CaM-binding kinesins confers Ca(2+)-CaM regulation, we fused the CBD of KCBP to the N or C terminus of a minus-end (non-claret disjunction) or C terminus of a plus-end (Drosophila kinesin) motor. Purified chimeric kinesins bound CaM in a Ca(2+)-dependent manner whereas non-claret disjunction, Drosophila kinesin, and KCBP that lack a CBD did not. As in the case of KCBP with CBD, the interaction of chimeric motors with MTs, as well as their MT-stimulated ATPase activity, was inhibited by Ca(2+)-CaM. The presence of a spacer between the motor and CBD did not alter Ca(2+)-CaM regulation. However, KCBP interaction with MTs and its MT-stimulated ATPase activity were not inhibited when the motor domain and CBD were added separately, suggesting that Ca(2+)-CaM regulation of CaM-binding motors occurs only when the CBD is attached to the motor domain. These results show that the fusion of the CBD to animal motors confers Ca(2+)-CaM regulation and suggest that the CBD functions as a modular domain in disrupting motor-MT interaction. Our data also support the hypothesis that CaM-binding kinesins may have evolved by addition of a CBD to a kinesin motor domain.     

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.