Involvement of Ca(2+)/calmodulin-dependent protein kinases in mycelial growth of the basidiomycetous mushroom, Coprinus cinereus

Although multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) are widely distributed in animal cells, the occurrence of CaM-kinases in the basidiomycetous mushroom has not previously been documented. When the extracts from various developmental stages from mycelia to the mature fruiting body of Coprinus cinereus were analyzed by Western blotting using Multi-PK antibodies, which had been generated to detect a wide variety of protein serine/threonine kinases (Ser/Thr kinases), a variety of stage-specific Ser/Thr kinases was detected. Calmodulin (CaM) overlay assay using digoxigenin-labeled CaM detected protein bands of 65 kDa, 58 kDa, 46 kDa, 42 kDa, and 38 kDa only in the presence of CaCl(2), suggesting that these bands were CaM-binding proteins. When the CaM-binding fraction was prepared from mycelial extract of C. cinereus by CaM-Sepharose and analyzed with Multi-PK antibodies, two major immunoreactive bands corresponding to 65 kDa and 46 kDa were detected. CaM-binding fraction, thus obtained, exhibited Ca(2+)/CaM-dependent protein kinase activity toward protein substrates such as histones. These CaM-kinases were found to be highly expressed in the actively growing mycelia, but not in the resting mycelial cells. Mycelial growth was enhanced by the addition of CaCl(2) in the culture media, but inhibited by the addition of EGTA or trifluoperazine, a potent CaM inhibitor. This suggested that CaM-dependent enzymes including CaM-kinases play crucial roles in mycelial growth of basidiomycete C. cinereus.     

Novel Ca/calmodulin-dependent protein kinase expressed in actively growing mycelia of the basidiomycetous mushroom Coprinus cinereus

We isolated cDNA clones for novel protein kinases by expression screening of a cDNA library from the basidiomycetous mushroom Coprinus cinereus. One of the isolated clones was found to encode a calmodulin (CaM)-binding protein consisting of 488 amino acid residues with a predicted molecular weight of 53,906, which we designated CoPK12. The amino acid sequence of the catalytic domain of CoPK12 showed 46% identity with those of rat Ca²⁺/CaM-dependent protein kinase (CaMK) I and CaMKIV. However, a striking difference between these kinases is that the critical Thr residue in the activating phosphorylation site of CaMKI/IV is replaced by a Glu residue at the identical position in CoPK12. As predicted from its primary sequence, CoPK12 was found to behave like an activated form of CaMKI phosphorylated by an upstream CaMK kinase, indicating that CoPK12 is a unique CaMK with different properties from those of the well-characterized CaMKI, II, and IV. CoPK12 was abundantly expressed in actively growing mycelia and phosphorylated various proteins, including endogenous substrates, in the presence of Ca²⁺/CaM. Treatment of mycelia of C. cinereus with KN-93, which was found to inhibit CoPK12, resulted in a significant reduction in growth rate of mycelia. These results suggest that CoPK12 is a new type of multifunctional CaMK expressed in C. cinereus, and that it may play an important role in the mycelial growth.     

CoPK32 is a novel stress-responsive protein kinase in the mushroom Coprinopsis cinerea

Background

In a previous study, we conducted an expression cloning screen of a cDNA library prepared from Coprinopsis cinerea mycelia using Multi-PK antibodies and detected a wide variety of Ser/Thr protein kinases. One of the isolated clones, CMZ032, was found to encode a putative Ser/Thr protein kinase designated CoPK32. In the present study, we investigated the biochemical properties and physiological significance of CoPK32.

Methods

CoPK32 was expressed in Escherichia coli, and its biochemical properties were examined. The effects of high osmotic stresses on the growth of C. cinerea and on the endogenous CoPK32 activity in mycelia were also examined.

Results

CoPK32 showed autophosphorylation activity and effectively phosphorylated exogenous protein substrates. CoPK32S, a splice variant that was 18 amino acids shorter than CoPK32, showed much lower protein kinase activity than CoPK32. The catalytic properties of CoPK32 deletion mutants suggested that the C-terminal region of CoPK32 was important for the kinase activity and recognition of substrates. CoPK32 was highly expressed in the actively growing region of the mycelial colony. When mycelia were stimulated by high osmotic stresses, endogenous CoPK32 was markedly activated and the mycelial growth was severely inhibited. The activation of CoPK32 activity by high osmotic stresses was abrogated by SB202190 or SB239063 as well-known inhibitors of p38 mitogen-activated protein kinase.

Conclusions

CoPK32 is involved in the stress response pathway in mycelia of C. cinerea in response to environmental stresses.

General significance

In C. cinerea, protein kinases such as CoPK32 play important roles in signal transduction pathways involved in stress responses.     

Extracellular calmodulin-binding proteins in plants: purification of a 21-kDa calmodulin-binding protein

A 21-kDa calmodulin (CaM)-binding protein and a 19-kDa calmodulin-binding protein were detected in 0.1 M CaCl₂ extracts of Angelica dahurica L. suspension-cultured cells and carrot (Daucus carota L.) suspension-cultured cells, respectively, using a biotinylated cauliflower CaM gel-overlay technique in the presence of 1 mM Ca²⁺. No bands, or very weak bands, were shown on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels overlayed with biotinylated cauliflower CaM when 1 mM Ca²⁺ was replaced by 5 mM EGTA, indicating that the binding of these two CaM-binding proteins to CaM was dependent on Ca²⁺. Less 21-kDa CaM-binding protein was found in culture medium of Angelica dahurica suspension cells; however, a 21-kDa protein was abundant in the cell wall. We believe that the 21-kDa CaM-binding protein is mainly in the cell wall of Angelica dahurica. Based on its reaction with periodic acid-Schiff (PAS) reagent, this 21-kDa protein would appear to be a glycoprotein. The 21-kDa CaM-binding protein was purified by a procedure including Sephadex G-100 gel filtration and CM-Sepharose cation-exchange column chromatography. The purity reached 91% according to gel scanning. The purified 21-kDa CaM-binding protein inhibited the activity of CaM-dependent NAD kinase and the degree of inhibition increased with augmentation of the 21-kDa protein, which appeared to be the typical characteristic of CaM-binding protein.     

Computational design of calmodulin mutants with up to 900-fold increase in binding specificity

Calmodulin (CaM) is a ubiquitous second messenger protein that regulates a variety of structurally and functionally diverse targets in response to changes in Ca²⁺ concentration. CaM-dependent protein kinase II (CaMKII) and calcineurin (CaN) are the prominent CaM targets that play an opposing role in many cellular functions including synaptic regulation. Since CaMKII and CaN compete for the available Ca²⁺/CaM, the differential affinity of these enzymes for CaM is crucial for achieving a balance in Ca²⁺ signaling. We used the computational protein design approach to modify CaM binding specificity for these two targets. Starting from the X-ray structure of CaM in complex with the CaM-binding domain of CaMKII, we optimized CaM interactions with CaMKII by introducing mutations into the CaM sequence. CaM optimization was performed with a protein design program, ORBIT, using a modified energy function that emphasized intermolecular interactions in the sequence selection procedure. Several CaM variants were experimentally constructed and tested for binding to the CaMKII and CaN peptides using the surface plasmon resonance technique. Most of our CaM mutants demonstrated small increase in affinity for the CaMKII peptide and substantial decrease in affinity for the CaN peptide compared to that of wild-type CaM. Our best CaM design exhibited an about 900-fold increase in binding specificity towards the CaMKII peptide, becoming the highest specificity switch achieved in any protein-protein interface through the computational protein design approach. Our results show that computational redesign of protein-protein interfaces becomes a reliable method for altering protein binding affinity and specificity.     

High molecular weight calmodulin-binding protein is phosphorylated by calmodulin-dependent protein kinase VI from bovine cardiac muscle

A high molecular weight calmodulin-binding protein (HMW CaMBP) from bovine heart cytosolic fraction was purified to apparent homogeneity. A novel CaM-dependent protein kinase was originally discovered when the total CaM-binding protein fraction from cardiac muscle was loaded on a gel filtration column. The CaM-dependent protein kinase was shown by gel filtration chromatography to have an apparent molecular mass of 36,000 daltons. The CaM-dependent protein kinase has been highly purified by sequential chromatography on DEAE-Sepharose C1 6B (to remove calmodulin), CaM-Sepharose 4B, phosphocellulose, Sepharose 6B gel filtration and Mono S column chromatographies. The highly purified protein kinase stoichiometrically phosphorylated the HMW CaMBP in a Ca²⁺/CaM-dependent manner. The phosphorylation resulted in the maximal incorporation of 1 mol of phosphate/mol of the HMW CaMBP. The distinct substrate specificity of this protein kinase indicates that it is not related to the known protein kinases (I, II, III, IV and V) that have been already characterized, therefore we would like to designate this novel kinase as a CaM-dependent protein kinase V1.     

Cellular Distribution of Calmodulin and Calmodulin-Binding Proteins in Vicia faba L.

The distribution of calmodulin (CaM) and CaM-binding proteins within Vicia faba was investigated. Both CaM and CaM-binding proteins were found to be differentially distributed among organs, tissues, and protoplast types. CaM levels, on a per protein basis, were found to be the highest in leaf epidermis, containing 3-fold higher levels of CaM than in total leaf. Similarly, guard cell and epidermal cell protoplasts were also found to have higher levels of CaM than mesophyll cell protoplasts. ¹²⁵I-CaM blot overlay assays were performed to qualitatively examine CaM-binding proteins in these protoplast types as well as in whole tissues and organs. CaM-binding proteins with M_r 52,000, 78,000, and 115,000 were common in all metabolically active plant parts. Unique CaM-binding protein bands were detected in guard cell protoplasts (M_r 39,000, 88,000), stems (M_r 45,000, 60,000, 64,000), and roots (M_r 62,000), suggesting the presence of specialized CaM-dependent processes in these cells and organs.     

Tyrosine kinase activity of a Ca2+/calmodulin-dependent protein kinase II catalytic fragment

A 30-kDa fragment of Ca²⁺/calmodulin-dependent protein kinase II (30K-CaMKII) is a constitutively active protein Ser/Thr kinase devoid of autophosphorylation activity. We have produced a chimeric enzyme of 30K-CaMKII (designated CX₄₀-30K-CaMKII), in which the N-terminal 40 amino acids of Xenopus Ca²⁺/calmodulin-dependent protein kinase I (CX₄₀) were fused to the N-terminal end of 30K-CaMKII. Although CX₄₀-30K-CaMKII exhibited essentially the same substrate specificity as 30K-CaMKII, it underwent significant autophosphorylation. Surprisingly, its autophosphorylation site was found to be Tyr-18 within the N-terminal CX₄₀ region of the fusion protein, although it did not show any Tyr kinase activity toward exogenous substrates. Several lines of evidence suggested that the autophosphorylation occurred via an intramolecular mechanism. These data suggest that even typical Ser/Thr kinases such as 30K-CaMKII can phosphorylate Tyr residues under certain conditions. The possible mechanism of the Tyr residue autophosphorylation is discussed.     

Interactions of the 18.5 kDA isoform of myelin basic protein with a2+-calmodulin: In vitro studies using gel shift assay

The interactions of the 18.5 kDa isoform of myelin basic protein (MBP) with calmodulin (CaM) in vitro have been investigated using glutaraldehyde or dithiobis[succinimidylpropionate] (DSP) cross-linking, and SDS-polyacrylamide gel electrophoresis. The following forms of MBP were used: the natural bovine C1 charge isomer (bMBP/C1) and a recombinant murine product (rmMBP), and their fragments generated by digestion with cathepsin D (EC 3.4.23.5). In physiological buffers (10 mM HEPES-NaOH, pH 7.4, 5 mM CaCl₂, 0.0035% glutaraldehyde; or 50 mM HEPES-NaOH, pH 7.4, 100 mM NaCl, 1 mM CaCl₂, 0.0035% DSP), MBP and CaM interacted primarily in a 1:1 molar ratio, consistent with previous studies that used 6 M urea, i.e. denaturing conditions. Moreover, the appearance of higher-order bands (not previously observed) suggested that the mechanism of interaction of the two proteins involved a series of relatively complex equilibria, resulting in 2:1 ratios of MBP to CaM. This observation would explain the cooperativity of association inferred from fluorescence studies [13]. Our results demonstrated further that the interaction involved the C-terminal domain of MBP, again in a primarily 1:1 molar ratio with CaM, consistent with our identification of a CaM-binding motif at the C-terminus.     

Distribution of calmodulin and calmodulin-binding proteins in bovine pituitary: association of myosin light chain kinase with pituitary secretory granule membranes

Calcium is necessary for secretion of pituitary hormones. Many of the biological effects of Ca²⁺ are mediated by the Ca²⁺-binding protein calmodulin (CaM), which interacts specifically with proteins regulated by the Ca²⁺-CaM complex. One of these proteins is myosin light chain kinase (MLCK), a Ca²⁺-calmodulin dependent enzyme that phosphorylates the regulatory light chains of myosin, and has been implicated in motile processes in both muscle and non-muscle tissues. We determined the content and distribution of CaM and CaM-binding proteins in bovine pituitary homogenates, and subcellular fractions including secretory granules and secretory granule membranes. CaM measured by radioimmunoassay was found in each fraction; although approximately one-half was in the cytosolic fraction, CaM was also associated with the plasma membrane and secretory granule fractions. CaM-binding proteins were identified by an ²⁵¹-CaM gel overlay technique and quantitated by densitometric analysis of the autoradiograms. Pituitary homogenates contained nine major CaM-binding proteins of 146, 131, 90, 64, 58, 56, 52, 31 and 22 kilodaltons (kDa). Binding to all the bands was specific, Cat+-sensitive, and displaceable with excess unlabeled CaM. Severe heat treatment (100°C, 15 min), which results in a 75% reduction in phosphodiesterase activation by CaM, markedly decreased ²⁵¹I-CaM binding to all protein bands. Secretory granule membranes showed enhancement for CaM-binding proteins with molecular weights of 184, 146, 131, 90, and 52000. A specific, affinity purified antibody to chicken gizzard MLCK bound to the 146 kDa band in homogenates, centrifugal subcellular fractions, and secretory granule membranes. No such binding was associated with the granule contents. The enrichment of MLCK and other CaM-binding proteins in pituitary secretory granule membranes suggests a possible role for CaM and/or CaM-binding proteins in granule membrane function and possibly exocytosis.     

An extracellular matrix, calmodulin-binding protein from Dictyostelium with EGF-like repeats that enhance cell motility

CyrA is a novel cysteine-rich protein with four EGFL repeats that was isolated using the calmodulin (CaM) binding overlay technique (CaMBOT), suggesting it is a CaM-binding protein (CaMBP). The full-length 63 kDa cyrA is cleaved into two major C-terminal fragments, cyrA-C45 and cyrA-C40. A putative CaM-binding domain was detected and both CaM-agarose binding and CaM immunoprecipitation verified that cyrA-C45 and cyrA-C40 each bind to CaM in both a Ca²⁺-dependent and -independent manner. cyrA-C45 was present continuously throughout growth and development but was secreted at high levels during the multicellular slug stage of Dictyostelium development. At this time, cyrA localizes to the extracellular matrix (ECM). ECM purification verified the presence of cyrA-C45. An 18 amino acid peptide (DdEGFL1) from the first EGFL repeat sequence of cyrA (EGFL1) that is present in both cyrA-C45 and -C40 enhances both random cell motility and cAMP-mediated chemotaxis. Here we reveal that the dose-dependent enhancement of motility by DdEGFL1 is related to the time of cell starvation. Addition of DdEGFL1 also inhibits cyrA proteolysis. The status of cyrA as an extracellular CaMBP was further clarified by the demonstration that CaM is secreted during development. Antagonism of CaM with W7 resulted in enhanced cyrA proteolysis suggesting a functional role for extracellular CaM in protecting CaMBPs from proteolysis. cyrA is the first extracellular CaMBP identified in Dictyostelium and since it is an ECM protein with EGF-like repeats that enhance cell motility and it likely also represents the first matricellular protein identified in a lower eukaryote.     

Characteristics of Ca2+/calmodulin- and Ca2+/phosphatidylserine-stimulated phosphoproteins in rat striatum

The characteristics of endogenous Ca²⁺/calmodulin (CaM)- and Ca²⁺/phosphatidylserine (PS)-stimulated phosphorylated proteins in the striatum of rat were partially determined and compared in this study. The Ca²⁺/CaM-dependent phosphoproteins were associated with serine and threonine residues. The sensitivity of these proteins for phosphorylation by Ca²⁺/CaM was not affected by pretreatment of tissue with Ca²⁺ chelating agent, EGTA or with non-ionic detergent, Triton X-114. Triton X-114 phase separation experiments revealed that these Ca²⁺/CaM-dependent phosphoproteins were partitioned in the detergent rich phase suggesting that they are integral proteins of the striatal membrane. On the other hand, the Ca²⁺/PS-dependent phosphorylated proteins were primarily associated with the serine residue. Phosphorylation of these proteins by Ca²⁺/PS were abolished after the treatment with EGTA or Triton X-114. These results suggest that Ca²⁺/PS-dependent striatal phosphoproteins are biochemically unstable in maintaining their state of phosphorylation.     

Phosphorylation of ribosomal protein S19 at Ser59 by CaM Kinase Iα

In order to examine the possible involvements of Ca²⁺/calmodulin-dependent protein kinases (CaM kinases) in the regulation of ribosomal functions, we tested the phosphorylation of rat ribosomal protein S19 (RPS19) by various CaM kinases in vitro . We found that CaM kinase Iα, but not CaM kinase Iβ1, Iβ2, II, or IV, robustly phosphorylated RPS19. From the consensus phosphorylation site sequence, Ser59, Ser90, and Thr124 were likely to be phosphorylated; therefore, we mutated each amino acid to alanine and found that the mutation of Ser59 to alanine strongly attenuated phosphorylation by CaM kinase Iα, suggesting that Ser59 was a major phosphorylation site. Furthermore, we produced a specific antibody against RPS19 phosphorylated at Ser59, and found that Ser59 was phosphorylated both in GT1-7 cells and rat brain. Phosphorylation of RPS19 in GT1-7 cells was inhibited by KN93, an inhibitor of CaM kinases. Immunoblot analysis after subcellular fractionation of rat brain demonstrated that phosphorylated RPS19 was present in 80S ribosomes. Phosphorylation of RPS19 by CaM kinase Iα augmented the interaction of RPS19 with the previously identified S19 binding protein. These results suggest that CaM kinase Iα regulates the functions of RPS19 through phosphorylation of Ser59.     

Characterization of a pathogen-induced calmodulin-binding protein: mapping of four Ca2+-dependent calmodulin-binding domains

Ca²⁺ and calmodulin (CaM), a key Ca²⁺ sensor in all eukaryotes, have been implicated in defense responses in plants. To elucidate the role of Ca²⁺ and CaM in defense signaling, we used ³⁵S-labeled CaM to screen expression libraries prepared from tissues that were either treated with an elicitor derived from Phytophthora megasperma or infected with Pseudomonas syringae pv. tabaci. Nineteen cDNAs that encode the same protein, pathogen-induced CaM-binding protein (PICBP), were isolated. The PICBP fusion proteins bound ³⁵S-CaM, horseradish peroxidase-labeled CaM and CaM-Sepharose in the presence of Ca²⁺ whereas EGTA, a Ca²⁺ chelator, abolished binding, confirming that PICBP binds CaM in a Ca²⁺-dependent manner. Using a series of bacterially expressed truncated versions of PICBP, four CaM-binding domains, with a potential CaM-binding consensus sequence of WSNLKKVILLKRFVKSL, were identified. The deduced PICBP protein sequence is rich in leucine residues and contains three classes of repeats. The PICBP gene is differentially expressed in tissues with the highest expression in stem. The expression of PICBP in Arabidopsis was induced in response to avirulent Pseudomonas syringae pv. tomato carrying avrRpm1. Furthermore, PICBP is constitutively expressed in the Arabidopsis accelerated cell death2-2 mutant. The expression of PICBP in bean leaves was also induced after inoculation with avirulent and non-pathogenic bacterial strains. In addition, the hrp1 mutant of Pseudomonas syringae pv. tabaci and inducers of plant defense such as salicylic acid, hydrogen peroxide and a fungal elicitor induced PICBP expression in bean. Our data suggest a role for PICBP in Ca²⁺-mediated defense signaling and cell-death. Furthermore, PICBP is the first identified CBP in eukaryotes with four Ca²⁺-dependent CaM-binding domains.     

Regulation of ryanodine receptors by sphingosylphosphorylcholine: Involvement of both calmodulin-dependent and -independent mechanisms

Sphingosylphosphorylcholine (SPC), a lipid mediator with putative second messenger functions, has been reported to regulate ryanodine receptors (RyRs), Ca²⁺ channels of the sarco/endoplasmic reticulum. RyRs are also regulated by the ubiquitous Ca²⁺ sensor calmodulin (CaM), and we have previously shown that SPC disrupts the complex of CaM and the peptide corresponding to the CaM-binding domain of the skeletal muscle Ca²⁺ release channel (RyR1). Here we report that SPC also displaces Ca²⁺-bound CaM from the intact RyR1, which we hypothesized might lead to channel activation by relieving the negative feedback Ca²⁺CaM exerts on the channel. We could not demonstrate such channel activation as we have found that SPC has a direct, CaM-independent inhibitory effect on channel activity, confirmed by both single channel measurements and [³H]ryanodine binding assays. In the presence of Ca²⁺CaM, however, the addition of SPC did not reduce [³H]ryanodine binding, which we could explain by assuming that the direct inhibitory action of the sphingolipid was negated by the simultaneous displacement of inhibitory Ca²⁺CaM. Additional experiments revealed that RyRs are unlikely to be responsible for SPC-elicited Ca²⁺ release from brain microsomes, and that SPC does not exert detergent-like effects on sarcoplasmic reticulum vesicles. We conclude that regulation of RyRs by SPC involves both CaM-dependent and -independent mechanisms, thus, the sphingolipid might play a physiological role in RyR regulation, but channel activation previously attributed to SPC is unlikely.     

Biochemical characterization of Ca2+/calmodulin dependent protein kinase from Candida albicans

A multifunctional Ca²⁺/calmodulin dependent protein kinase was purified approximately 650 fold from cytosolic extract of Candida albicans. The purified preparation gave a single band of 69 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis with its native molecular mass of 71 kDa suggesting that the enzyme is monomeric. Its activity was dependent on calcium, calmodulin and ATP when measured at saturating histone IIs concentration. The purified Ca²⁺/CaMPK was found to be autophosphorylated at serine residue(s) in the presence of Ca²⁺/calmodulin and enzyme stimulation was strongly inhibited by W-7 (CaM antagonist) and KN-62 (Ca²⁺/CaM dependent PK inhibitor). These results confirm that the purified enzyme is Ca²⁺/CaM dependent protein kinase of Candida albicans. The enzyme phosphorylated a number of exogenous and endogenous substrates in a Ca²⁺/calmodulin dependent manner suggesting that the enzyme is a multifunctional Ca²⁺/calmodulin-dependent protein kinase of Candida albicans.     

Phosphatidylinositol-4,5 bisphosphate (PIP2) inhibits apo-calmodulin binding to protein 4.1

Membrane skeletal protein 4.1R⁸⁰ plays a key role in regulation of erythrocyte plasticity. Protein 4.1R⁸⁰ interactions with transmembrane proteins, such as glycophorin C (GPC), are regulated by Ca²⁺-saturated calmodulin (Ca²⁺/CaM) through simultaneous binding to a short peptide (pep11; A²⁶⁴KKLWKVCVEHHTFFRL) and a serine residue (Ser¹⁸⁵), both located in the N-terminal 30 kDa FERM domain of 4.1R⁸⁰ (H·R30). We have previously demonstrated that CaM binding to H·R30 is Ca²⁺-independent and that CaM binding to H·R30 is responsible for the maintenance of H·R30 β-sheet structure. However, the mechanisms responsible for the regulation of CaM binding to H·R30 are still unknown. To investigate this, we took advantage of similarities and differences in the structure of Coracle, the Drosophila sp. homologue of human 4.1R⁸⁰, i.e. conservation of the pep11 sequence but substitution of the Ser¹⁸⁵ residue with an alanine residue. We show that the H·R30 homologue domain of Coracle, Cor30, also binds to CaM in a Ca²⁺-independent manner and that the Ca²⁺/CaM complex does not affect Cor30 binding to the transmembrane protein GPC. We also document that both H·R30 and Cor30 bind to phosphatidylinositol-4,5 bisphosphate (PIP₂) and other phospholipid species and that that PIP₂ inhibits Ca²⁺-free CaM but not Ca²⁺-saturated CaM binding to Cor30. We conclude that PIP₂ may play an important role as a modulator of apo-CaM binding to 4.1R⁸⁰ throughout evolution.