Activation by calmodulin of inositol-1,4,5-trisphosphate 3-kinase in guinea pig peritoneal macrophages

The activity of inositol-1,4,5-trisphosphate 3-kinase in the cytosol fraction of guinea pig macrophages was assayed with special reference to the dependence on the free Ca2+ concentration. The enzyme activity, as assessed by the production of inositol 1,3,4,5-tetrakisphosphate was reversibly activated by free Ca2+ concentrations ranging from 10(-7) to 10(-6)M. The calmodulin antagonists, W-7 and chlorpromazine, inhibited the Ca2+-activated enzyme activity in a dose-dependent fashion, thereby indicating that calmodulin may be involved in the activation by Ca2+. The content of calmodulin in the cytosol fraction (about 2.8 micrograms/mg of cytosol protein) was markedly reduced to less than 0.03 microgram/mg of proteins by subfractionation by ammonium sulfate, followed by an anion-exchange chromatography. The subfraction obtained by the chromatography showed no Ca2+ dependence in the enzyme activity, while an exogenous addition of calmodulin with 10(-6)M Ca2+ increased the enzyme activity. The enzyme activity was retained on a calmodulin-affinity column in the presence of Ca2+, and was eluted from the column by lowering the free Ca2+ concentration by adding ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. These results clearly indicate that calmodulin activates the inositol-1,4,5-trisphosphate 3-kinase activity.     

Ca2+/calmodulin binds and dissociates K-RasB from membrane

We have investigated the interaction of calmodulin (CaM) with Ras-p21 and the significance of this association. All Ras-p21 isoforms tested (H-, K-, and N-Ras) were detected in the particulate fraction of human platelets and MCF-7 cells, a human breast cancer cell line. In MCF-7 cells, H- and N-Ras were also detected in the cytosolic fraction. K-RasB from platelet and MCF-7 cell lysates was found to bind CaM in a Ca2+ -dependent but GTPgammaS-independent manner. The yeast two-hybrid analysis demonstrated that K-RasB binds to CaM in vivo. Incubation of isolated membranes from platelet and MCF-7 cells with CaM caused dissociation of only K-RasB from membranes in a Ca2+ -dependent manner. CaM antagonist, W7, inhibited dissociation of K-RasB. Addition of platelet or MCF-7 cytosol alone to isolated platelet membranes did not cause dissociation of K-RasB and only addition of exogenous CaM caused dissociation. The results suggest a potential role for Ca2+/CaM in the regulation of K-RasB function.     

Small Gtpase rab3A is associated with melanosomes in melanoma cells

Rab3A is a small guanosine triphosphate (GTP)-binding protein that has been recently implicated in intracellular vesicle transport and the secretion of neurotransmitters in neuronal cells. We demonstrate here that Rab3A is associated with melanosomes in pigment cells. Rab3A as well as Rabphilin3A, a putative target protein of Rab3A, were detected in the melanosome fraction, purified from B16 murine melanoma cells by sucrose density gradient ultracentrifugation. In contrast, Rab GDP dissociation inhibitor (GDI), a GDP/GTP exchange protein for Rab3A, was found in the cytosol fraction. Further studies using confocal laser scanning microscopy and immunoelectron microscopy revealed that immunoreactive Rab3A is localized in conjunction with the melanosomal membrane. These results suggest the possibility of involvement of Rab3A-Rabphilin3A complex, regulated by Rab GDI, in the intracellular transport of melanosomes in pigment cells.     

Dual role of DENN/MADD (Rab3GEP) in neurotransmission and neuroprotection

DENN/MADD is a component of a signalling protein complex that is localized to the cytosol and exerts multiple functions by using different binding partners. Human DENN/MADD is physically the same death-domain protein as rat Rab3 GDP/GTP exchange protein (Rab3GEP). DENN/MADD regulates the recycling of Rab3 small G proteins under normal conditions and has an essential role in Ca(2+)-dependent neurotransmitter release and exocytosis. It is also involved in blocking the apoptosis of neuronal cells under conditions of cytotoxic stress. Recent research supports an important role for DENN/MADD in neuroprotection: reduced endogenous DENN/MADD expression and enhanced pro-apoptotic signalling has been found in brains affected by Alzheimer's disease.     

Cloning and localization of Rab3 isoforms in bovine, rat, and human parathyroid glands

Rab3 proteins are small GTP-binding proteins known to play a role in regulated exocytosis processes. This study examines the expression of Rab3 mRNA and protein in bovine, rat and human parathyroid glands. mRNAs of several Rab3 isoforms were detected in bovine (Rab3A, Rab3B and Rab3C) and rat (Rab3A, Rab3B and Rab3D) parathyroid glands by RT-PCR and sequencing. Rab3A protein was detected in the cytosolic extract from bovine parathyroid gland by Western blotting using a monoclonal antibody for Rab3A. Rab3A protein was localized to parathyroid hormone-containing chief cells by immunohistochemical staining. Subcellular localization of Rab3A protein by immunogold electron microscopy revealed that the majority of Rab3A protein was not associated with dense-core vesicles, but localized in the cytosol of the chief cells. Altogether, our results demonstrate that Rab3 isoforms are expressed in parathyroid chief cells, suggesting that they may play a role in regulated exocytosis in these cells.     

Characterization of calcium-dependent membrane binding proteins of brain cortex.

Proteins of Mr 68 000, 34 000 and 32 000 were selectively extracted by EGTA from brain cortex. The three proteins that were extracted along with calmodulin were acidic, monomeric, and did not exhibit structural homology, as demonstrated by one-dimensional peptide mapping. The Mr-68 000 protein was purified to homogeneity and had a Stokes radius of 3.54 nm and S20,W value of 5.1S. Purified calmodulin, Mr-68 000 protein and two proteins of Mr 34 000 and Mr 32 000, interacted with the brain particulate fraction, with half-maximal binding occurring at 3.5 microM, 8.3 microM and 150 microM-Ca2+ respectively. Proteins were bound independently of each other and calmodulin. Pretreatment of the particulate fraction with trypsin prevented the Ca2+-dependent binding of calmodulin; however, the binding of the Mr-68 000 protein or the Mr-32 000 and -34 000 proteins was unaffected. The Mr-68 000 protein of bovine brain did not cross-react immunologically with Mr-67 000 calcimedin from chicken gizzard.     

Site-specific dephosphorylation of smooth muscle myosin light chain kinase by protein phosphatases 1 and 2A.

Smooth muscle myosin light chain kinase is phosphorylated at two sites (A and B) by different protein kinases. Phosphorylation at site A increases the concentration of Ca2+/calmodulin required for kinase activation. Diphosphorylated myosin light chain kinase was used to determine the site-specificity of several forms of protein serine/threonine phosphatase. These phosphatases readily dephosphorylated myosin light chain kinase in vitro and displayed differing specificities for the two phosphorylation sites. Type 2A protein phosphatase specifically dephosphorylated site A, and binding of Ca2+/calmodulin to the kinase had no effect on dephosphorylation. The purified catalytic subunit of type 1 protein phosphatase dephosphorylated both sites in the absence of Ca2+/calmodulin but only dephosphorylated site A in the presence of Ca2+/calmodulin. A protein phosphatase fraction was prepared from smooth muscle actomyosin by extraction with 80 mM MgCl2. On the basis of sensitivity to okadaic acid and inhibitor 2, this activity was composed of multiple protein phosphatases including type 1 activity. This phosphatase fraction dephosphorylated both sites in the absence of Ca2+/calmodulin. However, dephosphorylation of both sites A and B was completely blocked in the presence of Ca2+/calmodulin. These results indicate that two phosphorylation sites of myosin light chain kinase are dephosphorylated by multiple protein serine/threonine phosphatases with unique catalytic specificities.     

Free radical production from the aerobic oxidation of reduced pyridine nucleotides catalysed by phenazine derivatives

Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a (Ca2+ + Mg2+)-ATPase activity of about 10 nmol (min . mg)-1 and an ATP-dependent Ca2+ uptake of about 10 nmol (min . mg)-1. When incubated in the presence of Mg[gamma-32P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [32P]phosphate-labeled Ca2+ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca2+ or Ca2+ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of 125I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol (Mr = 94 000, 87 000, 60 000 and 43 000). Some minor calmodulin-binding proteins were enriched in the membrane fractions (Mr = 69 000, 57 000, 39 000 and 37 000). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca2+ uptake is essentially unaltered, while the Ca2+ capacity is diminished as a consequence of a doubling in the rate of Ca2+ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca2+ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 microM calmodulin result in increased levels of vesicle phosphorylation.     

Calcium/Calmodulin-Dependent Protein Kinase II in Squid Synaptosomes

The Ca2+/calmodulin (CaM)-dependent protein kinase II system in squid nervous tissue was investigated. The Ca2+/CaM-dependent protein kinase II was found to be very active in the synaptosome preparation from optic lobe, where it was associated with the high-speed particulate fraction. Incubation of the synaptosomal homogenate with calcium, calmodulin, magnesium, and ATP resulted in partial and reversible conversion of the Ca2+/CaM-dependent protein kinase II from its calcium-dependent form to a calcium-independent species. The magnitude of this conversion reaction could be increased by inclusion of the protein phosphatase inhibitor NaF or by substitution of adenosine 5'-O-(3-thiotriphosphate) for ATP. When [gamma-32P]ATP was used, proteins of 54 and 58 kilodaltons (kDa) as well as proteins greater than 100 kDa were rapidly 32P-labeled in a calcium-dependent manner. Major 125I-CaM binding proteins in the synaptosome membrane fraction were 38 and 54 kDa. The Ca2+/CaM-dependent protein kinase II was purified from the squid synaptosome and was shown to consist of 54- and 58-60-kDa subunits. The purified kinase, like Ca2+/CaM-dependent protein kinase II from rat brain, catalyzed autophosphorylation associated with formation of the calcium-independent form. These studies, characterizing the Ca2+/CaM-dependent protein kinase II in squid neural tissue, are supportive of the putative role of this kinase in regulating calcium-dependent synaptic functions.     

Adrenal Chromaffin Cell Calmodulin: Its Subcellular Distribution and Binding to Chromaffin Granule Membrane Proteins

Bovine adrenal medullae were homogenized in the presence or in the absence of EGTA and different subcellular fractions were prepared by differential and density gradient centrifugations. In the presence of the chelating agent, 69% of the total calmodulin, measured by radioimmunoassay, was present in the cytosol; the rest was bound to different membrane-containing fractions (nuclei, microsomal, and crude granule fraction). When the chelating agent was omitted, 43% of the calmodulin was present in the cytosol, the remaining calmodulin being membrane-bound. Further resolution of the crude granule fraction by sucrose density centrifugation demonstrated that the distribution of calmodulin in the density gradient was similar to the distribution of chromaffin granules rather than to that of mitochondria, Golgi elements, and lysosomes. In this case, there was also more calmodulin bound to chromaffin granules when EGTA was omitted from the density gradient. Experiments with 125I-calmodulin indicated the presence of high-affinity binding sites (KD = 1.3 X 10(-8) M; Bmax = 30 pmol/mg protein) for calmodulin in chromaffin granule membranes. Further, photoaffinity crosslinking experiments with 125I-calmodulin followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography indicated the presence of three calmodulin-binding polypeptide complexes (84,000; 41,000; and 38,000 daltons) in chromaffin granule membranes. These polypeptides were not labelled when either Ca2+ was omitted or an excess of nonradioactive calmodulin was present in the photolysis buffer, indicating the Ca2+ dependency and the specificity of the interaction. On the basis of the results described, it is suggested that the cellular levels of Ca2+ control the cellular distribution of calmodulin and its binding to specific chromaffin granule membrane proteins. Further, it is also suggested that the interactions between calmodulin and granule proteins might play a role in stimulus-secretion coupling.     

Characterization of the chromobindins. Soluble proteins that bind to the chromaffin granule membrane in the presence of Ca2+

A group of proteins that bind to the chromaffin granule membrane in the presence of Ca2+ has been isolated by affinity chromatography of bovine adrenal medullary cytosol on granule membranes coupled to Sepharose 4B. Twenty-two of these proteins were resolved into classes depending upon the Ca2+ concentration at which they were eluted from the affinity column (40 or 0.1 microM), upon their affinities for native granule membranes or for liposomes prepared from extracted granule lipids, and upon the requirement of seven of the proteins for ATP in the cytosol fraction and column buffers to promote binding. The molecular weights and isoelectric points of these proteins were determined by two-dimensional electrophoresis. Two of the granule-binding proteins were identified: synexin and calmodulin. Calmodulin was found to bind to seven specific granule membrane proteins after diffusion of 125I-labeled calmodulin into an acrylamide gel of membrane proteins separated by electrophoresis in the presence of sodium dodecyl sulfate. A phospholipid-activated protein kinase activity, possibly due to protein kinase C, was present in the granule-binding fraction. Two major granule-binding proteins were found to present a pattern in two-dimensional electrophoresis that was very similar to but shifted slightly toward the basic end of the gel from the pattern generated by light chains associated with clathrin in adrenal medullary coated vesicles. In the chromaffin cell, these proteins, by associating with the granule membrane in the presence of an increased cytosolic Ca2+ concentration, might play a variety of roles in the process of exocytosis.     

Calmodulin activates inositol 1,4,5-trisphosphate 3-kinase activity in pig aortic smooth muscle.

The effects of calmodulin (CaM) on inositol 1,4,5-trisphosphate (InsP3) 3-kinase activity in pig aortic smooth muscle were examined. The cytosol fraction of muscle cells, containing 1.2-2.0 micrograms of CaM/mg of cytosol protein (thus 0.12-0.2%, w/w), showed a Ca2+-dependent InsP3 3-kinase activity, and there was no further activation by exogenous addition of CaM purified from dog brain. (NH4)2SO4 fractionation of the cytosol fraction revealed that a 20-60%-satd.-(NH4)2SO4 fraction was rich in the enzyme activity, and the activity without exogenous CaM was still dependent on Ca2+, although the CaM content in this fraction was minute (0.013-0.016%, w/w). The kinase activity observed in the absence of exogenous CaM became insensitive to Ca2+ when a 20-60%-satd.-(NH4)2SO4 fraction was applied to a DEAE-cellulose column, but exogenous addition of CaM increased the enzyme activity from 80-120 to 450 pmol/min per mg of protein, with addition of 10 microM free Ca2+. A fraction separated by DEAE-cellulose chromatography was applied to a CaM affinity column. The kinase activity was retained on the column in the presence of Ca2+, and was eluted by lowering the free Ca2+ concentration by adding EGTA. These results directly show that CaM activates InsP3 3-kinase activity and the enzyme becomes sensitive to Ca2+.     

Properties of detergent-dispersed adenylate cyclase from cerebral cortex. Presence of an inhibitor protein

Adenylate cyclase was solubilized from washed particulate fraction of rabbit cerebral cortex with the nonionic detergent Lubrol 12A9 and subjected to either gel filtration on Ultrogel AcA 34 or chromatography on DEAE Bio-Gel A. By both procedures the enzyme was resolved into two components, one insensitive to guanyl 5'-yl imidodiphosphate [Gpp(NH)p] and NaF but stimulated by Ca2+ and calmodulin, and another that was sensitive to Gpp(NH)p and NaF but relatively insensitive to Ca2+ and calmodulin. The data support the possibility that two independent forms of adenylate cyclase exist in cerebral cortex, one regulated by guanine nucleotide regulatory protein and another by Ca2+-calmodulin. Fractions containing the guanylnucleotide-sensitive activity were found to contain a factor that inhibited basal and Ca2+-stimulated adenylate cyclase in the Ca2+-sensitive fraction. The inhibitor was inactivated by heating at 60 degrees C and by incubation with trypsin. Inhibition was not time-dependent, and it was not due to destruction of cAMP by phosphodiesterase or of ATP by ATPase. Inhibitory action was not reversed by calmodulin and therefore it does not appear to be a calmodulin binding protein. Sucrose density gradient sedimentation indicated a sedimentation coefficient of 4S for the inhibitor; by this technique it co-sedimented with the adenylate cyclase sensitive to Gpp(NH)p and NaF.     

Phorbol myristate acetate stimulates formation of phosphatidyl inositol 4-phosphate and phosphatidyl inositol 4,5-bisphosphate in human platelets

There are conflicting data in the literature as to whether or not the Ca2+ activation of phospholipase A2 is mediated by the calcium binding protein calmodulin. In the present study the membrane-bound phospholipase A2 enzymes in rat and human platelets were shown to be absolutely Ca2+ dependent but were not stimulated by the addition of calmodulin. A partially purified phospholipase A2 from rat platelet membrane, which contained little endogenous calmodulin, also was not stimulated by calmodulin addition. Both isolated and membrane-bound phospholipase A2 were inhibited by the non-specific calmodulin antagonist trifluoperazine but the inhibition was not overcome by adding calmodulin. There was thus no evidence from these studies that phospholipase A2 is calmodulin regulated.     

Subcellular distribution of protein kinase C/phorbol ester receptors in differentiating mouse keratinocytes

The activation of protein kinase C (PKC) by diacylglycerol or tumor promoters plays a pivotal role in signal transduction and subsequent activation of cellular processes. Since the activity of this enzyme is dependent on its immediate lipid domain, its relative distribution within the cell may be an important regulatory mechanism. We report here a relative decrease in PKC/phorbol ester receptor associated with the particulate fraction of mouse keratinocytes induced to differentiate by two separate systems. First, proliferating keratinocytes maintained in low Ca2+ (0.09 mM) serum-free medium were induced to differentiate rapidly by the addition of Ca2+ (1.8 mM). A 1.4-fold decrease in the percent of total phorbol receptor binding activity present in the particulate fraction and concomitant increase in binding in the cytosol fraction was evident 20 min after the Ca2+ addition. Second, in keratinocytes that differentiate over a 6 day cultivation period in serum-containing medium with Ca2+ concentration of 1.8 mM, a significant decrease in the percent of the phorbol receptor binding activity present in the particulate fraction was observed as the culture begins to differentiate on days 3 and 4. Maximal phorbol ester binding in the particulate fraction corresponded to the proliferative phase of the culture (day 2), while lower levels of PKC/phorbol ester binding to particulate fractions were noted during the early differentiative phase (days 3 and 4). Addition of the synthetic diacylglycerols 1-oleoyl-2-acetylglycerol or L-alpha-1,2 dioctanyl glycerol at 30 micrograms/ml to proliferating keratinocyte cultures induced a modest increase in two markers of terminal differentiation: cornified envelope formation and transglutaminase levels. These findings, taken together, support the hypothesis that PKC activation plays a role in the initial signalling events for keratinocyte differentiation.     

Effect of pentobarbital dependence on adenylate cyclase activity and calmodulin levels in rat cerebral cortex

The effect of calcium (Ca2+) on the adenylate cyclase activity and calmodulin level of cerebral cortex was determined in pentobarbital dependent rats and age matched controls. Female Sprague-Dawley rats were made dependent and maintained on pentobarbital by eating a mixture of pentobarbital and rat chow (350 mg pentobarbital/30 g chow). Ca2+ activated then inhibited the adenylate cyclase activity associated with a 20,000 X g particulate fraction from pentobarbital dependent and age matched control rats. The values for one-half maximal stimulation and inhibition by Ca2+ did not differ significantly in either cortical preparation. However, the ability of Ca2+ to activate adenylate cyclase from pentobarbital dependent animals was significantly decreased (p less than 0.05) when compared to control animals. Pentobarbital (10(-4) - 10(-3) added to particulate fractions from naive control rats did not alter the ability of Ca2+ to activate adenylate cyclase. The calmodulin levels in the particulate fraction from pentobarbital dependent animals (30.2 +/- 6.7 ng calmodulin/mg protein) did not differ significantly when compared to control (33.0 +/- 4.7 ng/mg). By contrast, the calmodulin levels (37.9 +/- 5.9 ng/mg) in the 20,000 X g supernatant from cortex of pentobarbital dependent animals was significantly greater than the level in the supernatant from control animals (28.6 +/- 2.6 ng/mg). The ability of forskolin, dopamine, GTP or forskolin plus GTP (all at a concentration of 100 microM) to activate adenylate cyclase was significantly decreased in particulate preparations from pentobarbital dependent animals. In summary, our data show that alterations in calmodulin levels and a decreased responsivity of adenylate cyclase occur in animals physically dependent on pentobarbital.     

Protein phosphatase 2B dephosphorylates twitchin, initiating the catch state of invertebrate smooth muscle

"Catch" is the state where some invertebrate muscles sustain high tension for long periods at low ATP hydrolysis rates. Physiological studies using muscle fibers have not yet fully provided the details of the initiation process of the catch state. The process was extensively studied by using an in vitro reconstitution assay with several phosphatase inhibitors. Actin filaments bound to thick filaments pretreated with the soluble protein fraction of muscle homogenate and Ca2+ (catch treatment) in the presence of MgATP at a low free Ca2+ concentration (the catch state). Catch treatment with > 50 microm okadaic acid, > 1 microm microcystin LR, 1 microm cyclosporin A, 1 microm FK506, or 0.2 mm calcineurin autoinhibitory peptide fragment produced almost no binding of the actin filaments, indicating protein phosphatase 2B (PP2B) was involved. Use of bovine calcineurin (PP2B) and its activator calmodulin instead of the soluble protein fraction initiated the catch state, indicating that only PP2B and calmodulin in the soluble protein fraction are essential for the initiation process. The initiation was reproduced with purified actin, myosin, twitchin, PP2B, and calmodulin. 32P autoradiography showed that only twitchin was dephosphorylated during the catch treatment with either the soluble protein fraction or bovine calcineurin and calmodulin. These results indicate that PP2B directly dephosphorylates twitchin and initiates the catch state and that no other component is required for the initiation process of the catch state.     

Differential mechanisms of translocation of protein kinase C to plasma membranes in activated human neutrophils

Three classes of activators of human neutrophils that induce the intracellular translocation of protein kinase C from the cytosol to the particulate fraction were compared for their effects on the properties of the particulate (membrane-bound) enzyme. In cells stimulated with 10 ng/ml of phorbol-12-myristate-13-acetate (PMA) the particulate enzyme is almost fully active in the absence of added Ca2+ or phospholipids and this activity is not released by the Ca2+-chelator EDTA. In contrast, binding of protein kinase C to the particulate fraction in cells treated with the chemotactic factor f-Met-Leu-Phe (fMLF) or with the ionophore A-23187 plus Ca2+ is observed only when the cells are lysed in the presence of 1 mM Ca2+. With these stimuli the particulate enzyme retains a nearly absolute requirement for Ca2+ and phospholipids. Thus only the full intercalation of protein kinase C caused by PMA, which is resistant to removal by chelators stabilizes an active form of protein kinase C in the neutrophil membrane. In confirmation of this conclusion, in isolated plasma membranes loaded with partially purified protein kinase C by incubation with 5 microM Ca2+ further incubation with PMA, but not with fMLF, caused a significant fraction of the bound PKC to become resistant to removal by chelators, and to be nearly fully active in the absence of added activators.     

Is Ca2+ -Calmodulin-Dependent Protein Phosphorylation in Rat Brain Modulated by Carboxylmethylation?

Calmodulin stimulation of protein kinase activity in calmodulin-depleted preparations of rat brain cytosol or synaptosomal membranes was attenuated by prior carboxylmethylation of the enzyme source with purified protein-O-carboxylmethyltransferase. Similarly, calmodulin stimulation of highly purified Ca2+-calmodulin-dependent protein kinase was reduced if the kinase was exposed to methylating conditions prior to addition of calmodulin. Biochemical and acidic sodium dodecyl sulfate-gel electrophoretic analyses indicated that all sources of protein kinase activity were substrates for methylation. The specific activity of methyl group incorporation into protein kinase increased with increasing purity of the preparation, reaching values of 1.72 pmol CH3/micrograms protein or 0.15-1.12 mol CH3/mol of holoenzyme. Analysis of ATP binding in cytosol with the use of the photoaffinity probe [32P]8-azido-ATP indicated that carboxylmethylation reduced ATP binding. These results suggest that carboxylmethylation of Ca2+-calmodulin protein kinase may modulate the activity of this enzyme in rat brain.