Site-specific mutagenesis of the alpha-helices of calmodulin. Effects of altering a charge cluster in the helix that links the two halves of calmodulin

Alteration of residues 82-84 in the alpha-helix that links the two halves of calmodulin results in a differential effect on activator activity. Previous studies (Lukas, T. J., Burgess, W. H., Prendergast, F. G., Lau, W., and Watterson, D. M. (1986) Biochemistry 25, 1458-1464) indicated the importance of positive charge clusters in the calmodulin-binding protein, myosin light chain kinase. This suggested the possible importance of complementary negative charge clusters in calmodulin. By using an efficient cassette mutagenesis approach and a synthetic calmodulin gene (Roberts, D. M., Crea, R., Malecha, M., Alvarado-Urbina, G., Chiarello, R. H., and Watterson, D. M. (1985) Biochemistry 24, 5090-5098), this possibility was directly addressed by engineering a new calmodulin, VU-8 calmodulin, in which the glutamate cluster at residues 82-84 in the synthetic gene product (VU-1 calmodulin) was replaced by three lysines. VU-8 calmodulin activated phosphodiesterase to the same maximal extent as VU-1 calmodulin, although there was an alteration in the concentration of calmodulin required for half-maximal stimulation. In contrast, myosin light chain kinase was activated to only 30% of maximal activity and NAD kinase was not activated. These results provide insight into the functional role of the unusual central helix structure found in the calmodulin family of proteins and indicate that different, although possibly overlapping, chemical complementarities are employed in the interaction between calmodulin and its various physiological targets.     

Affinity-based chromatography utilizing genetically engineered proteins. Interaction of Bordetella pertussis adenylate cyclase with calmodulin

An engineered calmodulin differs from vertebrate calmodulin in its ability to activate Bordetella pertussis adenylate cyclase, and this difference has been utilized as the basis for a new purification protocol for the adenylate cyclase. VU-8 calmodulin, in which 3 glutamic acid residues (residues 82-84) have been substituted with 3 lysine residues, has a 1000-fold lower apparent affinity for the adenylate cyclase, compared to vertebrate calmodulin, and decreased maximal activity. Because of the relatively calcium-independent nature of the interaction between calmodulin and the cyclase, the use of calmodulin-Sepharose conjugates in the purification of the cyclase requires the use of chaotropic agents for elution. However, when immobilized VU-8 calmodulin was tested as a calcium-dependent, affinity-based, adsorption chromatography step in the purification of the cyclase from culture media or bacterial extracts, the enzyme bound to the column in a calcium-dependent manner, and a nearly homogeneous enzyme was obtained in high yield. These results demonstrate the feasibility of using engineered calmodulins that have selective differences in activity for the rational design of rapid purification protocols for calmodulin-binding proteins as well as indicate the importance of the conserved negative charge cluster at residues 82-84 of calmodulin for activation of this cyclase.     

Trifluoperazine binding to mutant calmodulins

Trifluoperazine (TFP) binding by 14 calmodulins, including 12 produced by site-directed mutagenesis, was determined. While vertebrate calmodulin binds 4.2 +/- 0.2 equiv of TFP, Escherichia coli expressed but unmutated calmodulins bind about 5.0 +/- 0.5 equiv of TFP. The cause for this difference is not known. The E. coli expressed proteins consist of two different series expressed from different calmodulin genes, CaMI and SYNCAM. The wild-type genes code for proteins that differ by nine conservative amino acid substitutions. Both these calmodulins bind 5 equiv of TFP with similar affinities, thus none of these conservative substitutions has any additional effect on TFP binding. Some altered calmodulins (deletion of EE83-84 or SEEE81-84, changing DEE118-120----KKK, M124----I,E120----K, or E82----K) have no appreciable effect on TFP binding. Other mutations affect either the binding of one TFP (deletion of E84) or about two TFP (changing E84----K, EEE82-84----KKK, E67----A, DEQ6-8----KKK, or E11----K). The mutations that affect TFP binding are localized to three regions of calmodulin: The amino-terminal alpha-helix, the central helix between the two globular ends of calmodulin, and a calcium-binding site in the second calcium-binding domain. The results are consistent with each of these regions either directly participating in drug binding or involved structurally in maintaining or inducing the correct conformation for TFP binding in the amino-terminal half of calmodulin.     

Calcium/calmodulin-dependent protein kinase II. Characterization of distinct calmodulin binding and inhibitory domains

Regulatory domains of the multifunctional Ca2+/calmodulin-dependent protein kinase II were investigated utilizing synthetic peptides. These peptides were derived from the sequence between positions 281 and 319 as translated from the cDNA sequence of the rat brain 50-kDa subunit (Lin, C. R., Kapiloff, M. S., Durgerian, S., Tatemoto, K., Russo, A. F., Hanson, P., Schulman, H., and Rosenfeld, M. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5962-5966), which contain the putative calmodulin-binding region as well as potential autophosphorylation sites. Peptide 290 to 309 was found to be a potent calmodulin antagonist with an IC50 of 52 nM for inhibition of Ca2+/calmodulin-dependent protein kinase II. Neither truncation from the amino terminus (peptide 296-309) nor extension in the carboxyl-terminal direction (peptide 294-319) markedly affected calmodulin binding, whereas shortening the peptide from the carboxyl terminus (peptide 290-302) or from both ends (peptide 295-304) resulted in the elimination of this activity. Peptide 281-290 did not bind calmodulin, but was a good substrate for the enzyme, being phosphorylated at Thr-286. Several of the peptides inhibited the kinase in a partially competitive, substrate-directed manner, but were not themselves phosphorylated. These studies identify domains within Ca2+/calmodulin-dependent protein kinase II which may be involved in 1) inhibition of the kinase in the absence of calmodulin, 2) binding of calmodulin, and 3) the resulting activation. Additionally, it is suggested that phosphorylation of residues flanking these domains may be responsible for the known regulatory effects of autophosphorylation on the properties of the kinase.     

Myosin light chain kinase structure function analysis using bacterial expression

A 40-kDa fragment of chicken smooth muscle myosin light chain kinase was produced and partially purified from a bacterial expression system. This fragment exhibits calmodulin binding and substrate phosphorylation properties similar to those of the isolated chicken gizzard enzyme. A series of 3'-deletion mutants was prepared and used to produce proteins with the same NH2 terminus but with COOH termini varying over 180 amino acids. Results show that truncation of the enzyme at Ser-512 (based on the amino acid numbering system described for the partial cDNA clone by Guerriero, V., Jr., Russo, M. A., Olson, N. J., Putkey, J. A., and Means, A. R. (1986) Biochemistry 25, 8372-8381) does not alter calmodulin binding, calmodulin regulation, or enzymatic properties. Removal of an additional 5 residues from the COOH terminus completely inhibits calmodulin binding and results in an inactive kinase that can be fully activated by limited proteolysis. Site specific mutations within these 5 residues demonstrate that Gly-508 and Arg-509 are independently involved in calmodulin-dependent binding and activation of myosin light chain kinase. Truncation of the enzyme at residues within the protein kinase catalytic domain results in inactive protein that cannot be activated by proteolysis.     

Cloning of a Ca(2+)/calmodulin-dependent protein kinase gene from the filamentous fungus Arthrobotrys dactyloides

A Ca(2+)/calmodulin-dependent protein kinase (CaMK) gene was cloned and characterized from Arthrobotrys dactyloides, a nematode-trapping fungus. The resulting 373-amino-acid protein, FCaMK, has significant homology to mammalian CaMKs. FCaMK contains a serine/threonine kinase domain followed by a calmodulin-binding domain. The activation loop in FCaMK (amino acids 184-199) contains a phosphorylation site at threonine-188, which could be the target of a kinase activator. Truncated FCaMK mutants revealed that amino acids 296-324 are essential for calmodulin binding. An oligopeptide designed from residues 297-324 formed a stable peptide-calmodulin complex of 1:1 stoichiometry. Southern blot analysis detected a single copy of the fcamk gene, suggesting that FCaMK plays an important role in Ca(2+)/calmodulin signaling in A. dactyloides.     

Identification of a new 84/82 kDa calmodulin-binding protein, which also interacts with actin filaments, tubulin and spectrin, as synapsin I

A new 84/82 kDa calmodulin-binding protein, which also interacts with actin filaments, tubulin and spectrin, was purified from the bovine synaptosomal membrane. The binding of calmodulin to this protein was Ca2+-dependent, and was inhibited by trifluoperazine, the association constant being calculated to be 2.2 X 10(6) M-1. Maximally, 1 mol of calmodulin bound to 1 mol of the purified protein. This protein was phosphorylated by both kinase II (Ca2+- and calmodulin-dependent kinase) and cyclic AMP-dependent kinase. In addition, antibody against this protein was demonstrated to have an immunological crossreactivity with synapsin I in the synaptosomal membrane.     

Electrostatic attraction by surface charge does not contribute to the catalytic efficiency of acetylcholinesterase.

Acetylcholinesterases (AChEs) are characterized by a high net negative charge and by an uneven surface charge distribution, giving rise to a negative electrostatic potential extending over most of the molecular surface. To evaluate the contribution of these electrostatic properties to the catalytic efficiency, 20 single- and multiple-site mutants of human AChE were generated by replacing up to seven acidic residues, vicinal to the rim of the active-center gorge (Glu84, Glu285, Glu292, Asp349, Glu358, Glu389 and Asp390), by neutral amino acids. Progressive simulated replacement of these charged residues results in a gradual decrease of the negative electrostatic potential which is essentially eliminated by neutralizing six or seven charges. In marked contrast to the shrinking of the electrostatic potential, the corresponding mutations had no significant effect on the apparent bimolecular rate constants of hydrolysis for charged and non-charged substrates, or on the Ki value for a charged active center inhibitor. Moreover, the kcat values for all 20 mutants are essentially identical to that of the wild type enzyme, and the apparent bimolecular rate constants show a moderate dependence on the ionic strength, which is invariant for all the enzymes examined. These findings suggest that the surface electrostatic properties of AChE do not contribute to the catalytic rate, that this rate is probably not diffusion-controlled and that long-range electrostatic interactions play no role in stabilization of the transition states of the catalytic process.     

Electric charge balance mechanism of extended soluble proteins

Extended proteins such as calmodulin and troponin C have two globular terminal domains linked by a central region that is exposed to water and often acts as a function-regulating element. The mechanisms that stabilize the tertiary structure of extended proteins appear to differ greatly from those of globular proteins. Identifying such differences in physical properties of amino acid sequences between extended proteins and globular proteins can provide clues useful for identification of extended proteins from complete genomes including orphan sequences. In the present study, we examined the structure and amino acid sequence of extended proteins. We found that extended proteins have a large net electric charge, high charge density, and an even balance of charge between the terminal domains, indicating that electrostatic interaction is a dominant factor in stabilization of extended proteins. Additionally, the central domain exposed to water contained many amphiphilic residues. Extended proteins can be identified from these physical properties of the tertiary structure, which can be deduced from the amino acid sequence. Analysis of physical properties of amino acid sequences can provide clues to the mechanism of protein folding. Also, structural changes in extended proteins may be caused by formation of molecular complexes. Long-range effects of electrostatic interactions also appear to play important roles in structural changes of extended proteins.     

Modulation of HERG gating by a charge cluster in the N-terminal proximal domain

Human ether-a-go-go-related gene (HERG) potassium channels contribute to the repolarization of the cardiac action potential and display unique gating properties with slow activation and fast inactivation kinetics. Deletions in the N-terminal 'proximal' domain (residues 135-366) have been shown to induce hyperpolarizing shifts in the voltage dependence of activation, suggesting that it modulates activation. However, we did not observe a hyperpolarizing shift with a subtotal deletion designed to preserve the local charge distribution, and other deletions narrowed the region to the KIKER containing sequence 362-372. Replacing the positively charged residues of this sequence by negative ones (EIEEE) resulted in a -45 mV shift of the voltage dependence of activation. The shifts were intermediate for individual charge reversals, whereas E365R resulted in a positive shift. Furthermore, the shifts in the voltage dependence were strongly correlated with the net charge of the KIKER region. The apparent speeding of the activation was attributable to the shifted voltage dependence of activation. Additionally, the introduction of negative charges accelerated the intermediate voltage-independent forward rate constant. We propose that the modulatory effects of the proximal domain on HERG gating are largely electrostatic, localized to the charged KIKER sequence.     

The receptor-associated protein (RAP) binds calmodulin and is phosphorylated by calmodulin-dependent kinase II.

The receptor-associated protein, RAP, is an intracellular protein that may function as a chaperone for the LDL-receptor family receptors. Here we report calmodulin as the first identified RAP binding protein outside of the LDL-receptor family members. We demonstrate that RAP binds calmodulin in a Ca2+- and pH-dependent manner characteristic of calmodulin-dependent enzymes, and present evidence that RAP is a substrate for calmodulin-dependent enzymes. Thus, CaM-kinase II and calcineurin readily phosphorylate and dephosphorylate, respectively, serine residues in RAP, and in the individual RAP domains D2 (amino acids 113-218) and D3 (amino acids 219-323) which both contain sites for CaM-kinase II-mediated phosphorylation and for calmodulin binding. In addition, we provide evidence that RAP is phosphorylated by other kinases such as casein kinase II. Studies of 32[ortho]P-labelled cell cultures demonstrate that RAP is phosphorylated in vivo. Our results suggest that RAP may have hitherto unknown functions implicating phosphorylation and calmodulin-mediated modulation.     

Smooth muscle myosin light chain kinase. Amino acid sequence at the site phosphorylated by adenosine cyclic 3',5'-phosphate-dependent protein kinase whether or not calmodulin is bound

Turkey gizzard smooth muscle myosin light chain kinase is a calmodulin-dependent enzyme containing 2 serine residues that can be phosphorylated by cAMP-dependent protein kinase. One of these sites can be phosphorylated only when calmodulin is not bound to the enzyme; the amino acid sequence around this site has been reported recently (Lukas, T. J., Burgess, W. H., Prendergast, F. G., Lau, W., and Watterson, D. M. (1986) Biochemistry 25, 1458-1464). Here we report the sequence around the site that is phosphorylated by cAMP-dependent protein kinase whether or not calmodulin is bound: Lys-Ala-Ser(P)-Gly-Ser-Ser-Pro-Thr-Ser-Pro-Ile-Asn-Ala-Asp-Lys-Val-Glu-A sn-Glu- . This sequence conforms to the previously defined criteria for substrates of cAMP-dependent protein kinase.     

A site-directed mutagenesis study of yeast calmodulin

A site-directed mutagenesis study was carried out in order to understand the regulatory mechanism of calmodulin. We started from the yeast (Saccharomyces cerevisiae) calmodulin gene since it has many differences in amino acid sequence and inferior functional properties compared with the vertebrate calmodulin. Recombinant yeast calmodulins were generated in Escherichia coli transformed by constructed expression plasmids. Three recombinant calmodulins were obtained. The first two were YCM61G, in which the Ca2(+)-binding site 2 (the four Ca2(+)-binding EF-hand structures in calmodulin were numbered from the N-terminus) was converted to the same as that in vertebrate calmodulin, and YCM delta 132-148, in which the C-terminal half sequence of site 4 was deleted. These two recombinant calmodulins had the same maximum Ca2+ binding (3 mol/mol) as yeast calmodulin, which indicates that site 4 of yeast calmodulin was the one losing Ca2+ binding capacity. YCM delta 132-148 could not activate target enzymes, whereas its Ca2+ binding profile was similar to those of yeast calmodulin and YCM61G. Therefore, the structure in site 4 which cannot bind Ca2+ is indispensable for the regulatory function of yeast calmodulin. The complete regulatory function of vertebrate calmodulin can be attained by the combination of 4 Ca2+ binding structures. The negative charge cluster in the central alpha-helix region is suggested to stabilize the active conformation of calmodulin, since the third yeast calmodulin mutant, YCM83E, which had the negative charge cluster, increased the maximum activation of myosin light chain kinase.     

Characterization of the calmodulin-binding and catalytic domains in skeletal muscle myosin light chain kinase

Limited proteolysis has been utilized to study the structural organization of rabbit skeletal muscle myosin light chain kinase. The enzyme (Mr approximately 89,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) consists of an amino-terminal, protease-susceptible region of unidentified function and a carboxyl-terminal, protease-resistant region of Mr approximately 40,000 containing the catalytic and calmodulin-binding domains. Partial digestion with trypsin produced an intermediate 56,000-dalton fragment and a stable 38,000-dalton fragment, both of which were catalytically active and calmodulin-dependent. Chymotryptic digestion yielded three catalytically active fragments of about 37,000, 36,000, and 35,000 daltons. The Mr = 37,000 fragment was calmodulin-dependent with an apparent affinity equivalent to that of the native enzyme (approximately 1 nM). The 36,000-dalton fragment was also calmodulin-dependent but had a approximately 200-fold lower apparent affinity. The Mr = 35,000 fragment was calmodulin-independent. These three chymotryptic fragments, had identical amino termini. Nineteen residues were missing from the carboxyl terminus of the calmodulin-independent chymotryptic fragment whereas only 8 or 9 carboxyl-terminal residues were missing from the calmodulin-dependent tryptic fragments. These results suggest that the 11-residue sequence (IAVSAANRFKK) in the carboxyl-terminal region of myosin light chain kinase contributes directly to the binding of calmodulin. This conclusion is in accord with data (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, K., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3187-3191) that the carboxyl-terminal, 27-residue CNBr peptide of the native enzyme shows Ca2+-dependent, high affinity binding to calmodulin and that similar calmodulin-binding activity, although detectable in unfractionated CNBr digests of calmodulin-dependent enzyme forms, is much reduced in a CNBr digest of the calmodulin-independent, Mr = 35,000 chymotryptic fragment.     

Purification and properties of a multifunctional calcium/calmodulin-dependent protein kinase from rat pancreas

A calcium/calmodulin-dependent protein kinase (Ca/calmodulin protein kinase) was purified from rat pancreas using hydrophobic chromatography followed by gel filtration and affinity chromatography. Ca/calmodulin protein kinase from pancreas resembled previously described multifunctional Ca/calmodulin protein kinases from other tissues with respect to substrate specificity, autophosphorylation on serine and threonine residues, and catalytic and hydrodynamic properties. While Ca/calmodulin protein kinase from other tissues contains subunits of 53-60 kDa with variable proportions of a smaller 50-52 kDa subunit, pancreatic Ca/calmodulin protein kinase was found to contain a single component of 51 kDa. Experiments mixing brain Ca/calmodulin protein kinase with pancreatic homogenate suggest that the absence of a larger subunit in the pancreatic Ca/calmodulin protein kinase is not due to proteolytic degradation during enzyme preparation. Ca/calmodulin protein kinase binding to 125I-labeled calmodulin in solution was demonstrated using the photoaffinity cross-linker, N-hydroxysuccinimidyl-4-azidobenzoate. 125I-labeled calmodulin binding to Ca/calmodulin protein kinase was also demonstrated using filters containing Ca/calmodulin protein kinase transferred from polyacrylamide gels after two-dimensional gel electrophoresis. Finally, the ribosomal substrate for Ca/calmodulin protein kinase was identified as the ribosomal protein, S6. The purification procedure presented in this study promises to be useful in characterizing Ca/calmodulin protein kinase in other tissues and in clarifying the role of these enzymes in cellular function.     

Immunohistochemical detection of calmodulin and calmodulin-dependent protein kinase II in the mouse testis

We reported previously that in mouse testis calmodulin-dependent protein phosphatase (calcineurin) is localised in the nuclei of round and elongating spermatids (Cell Tissue Res. 1995; 281: 273-81). In this study, we studied the immunohistochemical localisation of calcium/calmodulin-dependent protein kinase (CaM kinase II) using antibodies against CaM kinase IIgamma from chicken gizzard and specific antibodies raised against the amino acid sequence Ileu480-Ala493 of this enzyme, and compared it with the distribution of calmodulin. Indirect immunofluorescence was most concentrated in early spermatocytes and localised in the outermost layer of seminiferous tubules where the calmodulin level was relatively low. Measurements of immuno-gold particle densities on electron micrographs revealed that CaM kinase II is transiently increased in the nucleus of zygotene spermatocytes. These observations suggest the involvement of CaM kinase II in the meiotic chromosomal pairing process. An extremely high concentration of calmodulin in spermatogenic cells undergoing meiosis may not be directly related to activation of calmodulin-dependent kinases and phosphatases.     

Ca2+– and Calmodulin-Dependent Phosphorylation of Microtubule-Associated Protein 2 and t Factor, and Inhibition of Microtubule Assembly

Microtubule-associated proteins (MAPs) were phosphorylated by a Ca2+- and calmodulin-dependent protein kinase from rat brain cytosol. The maximal amount of phosphate incorporated into MAPs was 25 nmol of phosphate/mg protein. A Ka value of the enzyme for calmodulin was 57.0 nM, with MAPs as substrates. Among MAPs, MAP2 and tau factor were phosphorylated in a Ca2+- and calmodulin-dependent manner. The phosphorylation of MAPs led to an inhibition of microtubule assembly in accordance with its degree. This reaction was dependent on addition of the enzyme, Ca2+, and calmodulin, and had a greater effect on the initial rate of microtubule assembly rather than on the final extent. The critical tubulin concentration for microtubule assembly was unchanged by the MAPs phosphorylation. Therefore assembly and disassembly of brain microtubule are regulated by the Ca2+- and calmodulin-dependent protein kinase that requires only a nanomolar concentration of calmodulin for activation.     

Phosphorylation of Glial Fibrillary Acidic Protein and Vimentin by Cytoskeletal-Associated Intermediate Filament Protein Kinase Activity in Astrocytes

These studies describe a cytoskeletal-associated protein kinase activity in astrocytes that phosphorylated the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin and that appeared to be distinct from protein kinase C (PK-C) and the cyclic AMP-dependent protein kinase (PK-A). The cytoskeletal-associated kinase activity phosphorylated intermediate filament proteins in the presence of 10 mM MgCl2 and produced an even greater increase in 32P incorporation into these proteins in the presence of calcium/calmodulin. Tryptic peptide mapping of phosphorylated intermediate filament proteins showed that the intermediate filament protein kinase activity produced unique phosphopeptide maps, in both the presence and the absence of calcium/calmodulin, as compared to that of PK-C and PK-A, although there were some common sites of phosphorylation among the kinases. In addition, it was determined that the intermediate filament protein kinase activity phosphorylated both serine and threonine residues of the intermediate filament proteins, vimentin and GFAP. However, the relative proportion of serine and threonine residues phosphorylated varied depending on the presence or absence of calcium/calmodulin. The magnesium-dependent activity produced the highest proportion of threonine phosphorylation, suggesting that the calcium/calmodulin-dependent kinase activity acts mainly at serine residues. PK-A and PK-C phosphorylated mainly serine residues. Also, the intermediate filament protein kinase activity phosphorylated both the N-and the C-terminal domains of vimentin and the N-terminal domain of GFAP. In contrast, both PK-C and PK-A are known to phosphorylate the N-terminal domains of both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calmodulin and Ca2+- and Calmodulin-Dependent Protein Kinase in Rat Anterior Pituitary Gland

Calmodulin and Ca2+- and calmodulin-dependent protein kinase were identified in the rat anterior pituitary gland. The concentration of calmodulin was 1.18 +/- 0.11 microgram/mg protein (n = 7) in the cytosol fraction. The calmodulin of the anterior pituitary gland co-migrated with brain calmodulin on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The Ka value of the partially purified enzyme for Ca2+ was 3.3 microM in the presence of 0.30 microM calmodulin. Trifluoperazine and chlorpromazine, calmodulin-interacting agents, inhibited enzyme activity, with Ki values of 1.3 and 2.6 X 10(-5) M, respectively. The enzyme was resolved into two peaks of activity, with sedimentation coefficients of 5.5 S and 16.5 S, by sucrose density gradient centrifugation. At least nine proteins were phosphorylated by the enzyme in a Ca2+- and calmodulin-dependent manner. In light of these results, the possibility that calmodulin and the calmodulin-activatable protein kinase system are involved in the mediation of the Ca2+ effect on hormone release from the anterior pituitary gland must be given consideration.     

Inhibitory effect of regucalcin on Ca2+/calmodulin-dependent protein kinase activity in rat renal cortex cytosol

The effect of regucalcin on Ca²⁺/calmodulin-dependent protein kinase activity in the cytosol of rat renal cortex was investigated. Regucalcin is a calcium-binding protein which exists in rat liver and renal cortex. Protein kinase activity in renal cortex cytosol was markedly increased by the addition of CaCl₂ (0.5 mM) plus calmodulin (10 µg/ml) in the enzyme reaction mixture. This increase was completely prevented by the addition of trifluoperazine (25 µM), an antagonist of calmodulin. The cytosolic Ca²⁺/calmodulin- dependent protein kinase activity was clearly inhibited by the addition of regucalcin; an appreciable effect of regucalcin was seen at 0.01 µM. The cytosolic Ca²⁺/calmodulin-dependent protein kinase activity was fairly increased by increasing concentrations of added Ca²⁺ (100-1000 µM). This increase was markedly blocked by the presence of regucalcin (0.1 µM). The inhibitory effect of regucalcin on the protein kinase activity was also seen with varying concentrations of calmodulin (2-20 µg/ml). These results demonstrate that regucalcin can regulate Ca²⁺/calmodulin-dependent protein kinase activity in renal cortex cells.