Molecular and Cellular Biochemistry 252: 183–191, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.
183
Biochemical characterization of Ca2+/calmodulin dependent protein kinase from Candida albicans Navneet Kaur Dhillon, Sadhna Sharma and G.K. Khuller Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India Received 31 September 2002; accepted 10 February 2003
Abstract 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. (Mol Cell Biochem 252: 183–191, 2003) Key words: Ca2+, calmodulin, Ca2+/calmodulin dependent protein kinase, Candida albicans
Introduction Calcium is one of the predominant intracellular second messengers for the activation and modulation of biochemical and physiological processes in both prokaryotes and eukaryotes. Many of its functions are accomplished through interaction of calcium with calmodulin (CaM), a ubiquitous, heat stable, Ca2+ binding protein [1]. The Ca2+/CaM complex act in part through Ca2+/CaM dependent protein kinase(s), which are capable of phosphorylating and coordinately modifying the activities of several regulatory enzymes and structural proteins. CaM kinases thus play a pivotal role in cellular responses to stimuli that are mediated by calcium [2, 3]. Candida albicans is the most frequently isolated fungal pathogen in humans. The ability to switch between the yeast and the filamentous form has been postulated to contribute to the virulence of this organism [4]. Chlorpromazine (CPZ), a known calmodulin antagonist and trifluoperazine (TFP), a
calmodulin inhibitor [5, 6] have been shown to block germ tube formation by Candida albicans, thus suggesting the involvement of Ca2+/calmodulin dependent regulation of its morphogenesis. Besides morphogenesis, Ca2+ and calmodulin are known to play a role in yeast cell division cycle and nuclear division [7, 8]. Recently, our laboratory has demonstrated cell cycle arrest in the presence of CPZ and TFP consistent with the involvement of calmodulin in Candida albicans cell cycle [9]. Ca2+/calmodulin may affect phosphorylation of specific proteins through Ca2+/CaM dependent protein kinases, which may result in differential gene expression, that would in turn result in bud to hypha transition or cell division cycle in C. albicans. Identification and purification of putative protein kinases might unravel the signal transduction pathway involved in morphogenesis. Therefore, in this study, we have purified the Ca2+/CaM dependent protein kinase to homogeneity from Candida albicans.
Address for offprints: G.K. Khuller, Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh 160 012, India (E-mails:
[email protected];
[email protected])
184
Materials and methods Materials [γ-32p]-ATP was purchased from Bhabha Atomic Research Centre (Bombay, India), DEAE-cellulose, W-7, KN-62, H7, PKA-inhibitor-(5-24)-peptide (PKI), PMSF, leupeptin, pepstatin A, MOPS [3-(N-morpholino) propane sulfonic acid), EDTA, EGTA, ATP, casein, kemptide, autocamtide-2, phosvitin, syntide-2, histone IIs, BSA and molecular weight standards for gel electrophoresis were purchased from Sigma (St. Louis, MO, USA). Sephacryl S-300 was purchased from Pharmacia Biotech. Phosphocellulose paper (P81) was obtained from Whatman International (UK). Centricon 5 concentrators and ultrafiltration assembly (YM10) were from Amicon, USA. All other reagents were of highest available quality.
Organism and growth conditions Candida albicans ATCC 3153 obtained from Mycological Reference Laboratory (School of Hygiene and Tropical Medicine, London) was maintained on Sabouraud’s dextrose broth and was grown as shaking culture using mineral salt solution [10].
Ca2+/CaM dependent protein kinase assay The Ca2+/CaM-dependent protein kinase assay system contained in a final volume of 50 µl; 50 mM MOPS (pH 7.0), 10 mM MgCl2, 0.25 mg/ml BSA, 1.0 mg/ml histone IIs, 100 µM ATP containing [γ-32P] ATP (~ 100 cpm/pmole) and a suitable amount of enzyme in the presence of 50 µM CaCl2 and 1 µM CaM or in the presence of 1 mM EGTA. After incubation at 30°C for 5 min, the reaction was terminated by spotting a 25 µl aliquot of the mixture onto a phosphocellulose paper P81 (Whatman) and immediately placed in 75 mM phosphoric acid. The incorporated 32P was measured according to the method of Roskoski [11].
Purification of Ca2+/calmodulin-dependent protein kinase (CaMPK) Step 1: Preparation of soluble extract All steps were performed at – 4°C. Cells were resuspended in sonication buffer (20 µM Tris HCl, 1 mM DTT, 1 mM EGTA, 1 mM EDTA, 4 mM PMSF, pH 7.6) and then sonicated for 20 min with 1 min pause between each cycle for 1 min. The homogenate was centrifuged at 30,000g for 20 min.
The supernatant (crude) was then ultracentrifuged at 105,000 × g for 1 h to obtain cytosolic extract. Step II: Ammonium sulfate precipitation Ammonium sulfate was added to the cytosolic extract to a final concentration of 60%, stirred slowly and pH 7.6 was maintained by adding ammonium hydroxide (1:1) solution drop-wise. After keeping for 60 min at 4°C, the precipitate was collected by centrifugation at 27,000 × g for 20 min. The pellet was dissolved in 20 ml of buffer A (20 mM Tris HCl, 1 mM DTT, 1 mM EGTA, 0.02% Tween 20, 4 mM PMSF, 10% glycerol, pH 7.6) and then dialysed against three changes of 0.5 L of the same buffer for 12–16 h. Step III: DEAE cellulose chromatography The dialysate was clarified by centrifugation at 27,000 × g for 30 min and then applied to DEAE-cellulose column (1.4 × 30 cm), previously equilibrated with buffer A. The column was washed with five column volumes of equilibration buffer and then eluted with a linear NaCl gradient from 0–0.6 M (160 ml each) at a flow rate of 20 ml/h. Fractions of 4.0 ml were collected and monitored for calmodulin-dependent protein kinase activity. Step IV: Sephacryl S-300 gel permeation chromatography The fractions eluted from the DEAE cellulose column corresponding to the CaMPK activity were pooled, concentrated by amicon ultrafiltration assembly and loaded on pre-equilibrated Sephacryl S-300 gel filtration column (2 × 74 cm). The column was eluted with buffer A containing 200 mM NaCl at a flow rate of 30 ml/h and fractions of 5 ml were collected. Active fractions were examined for its purity on SDS-polyacrylamide gel electrophoresis and purified fractions were pooled and stored at –80°C.
Determination of molecular weight under non-denaturing conditions Gel filtration was carried out on Superdex-75 column (3.2 × 300 mm) attached to SMART Amersham Pharmacia FPLC system, equilibrated with buffer A. Purified CaM kinase or standard proteins were applied and eluted with the same buffer at the flow rate of 0.1 ml/min. Bovine serum albumin (66 kDa), ovalbumin (44 kDa), α-chymotrypsin (25 kDa), cytochrome c (12.4 kDa) and insulin (5.7 kDa) were used as standard proteins. Elution volume (Ve) was determined by reading the absorption maxima at 280 nm. The void volume (Vo) of the column was determined using Blue Dextran 2000. The plot of Ve/Vo vs. log molecular weight was then used to determine the native molecular mass of purified CaMPK from Candida.
185 Autophosphorylation of CaMPK
Results
The autophosphorylation of 10 µg purified CaMPK was carried out in a final volume of 50 µl under standard assay conditions, except that histone IIs was omitted and [γ-32P] ATP of higher specific activity (1000 cpm/pmol) was used. After incubation at 30°C for 30 min, 12.5 µl of SDS-PAGE sample buffer was added to the assay mixture and immediately boiled for 2 min. The sample was then subjected to SDS-PAGE and 32P incorporation was visualized by autoradiography
Purification of Ca2+/CaM kinase
Phosphorylation of endogenous substrates Candida albicans cells were sonicated in buffer A and centrifuged at 15,000 g for 15 min. The supernatant was used as substrate after incubation at 55°C for 60 min. Phosphorylation of the crude extract was carried out under standard assay conditions except that the heated extract was added instead of histone IIs as substrate and [γ-32P] ATP of higher specific activity (1000–1500 cpm/mol) was used and subjected to SDS-PAGE after boiling for 2–3 min with Laemmli’s sample buffer. The gels were dried and subjected to autoradiography.
Phosphoamino acid analysis Purified enzyme was incubated in the absence and presence of crude cell extract under standard phosphorylation conditions. After 30 min of incubation, the reaction was stopped by the addition of 10% ice-cold TCA. The precipitate obtained after centrifugation was washed twice with ice-cold acetone and hydrolyzed with 6 N HCl for 7 h at 110°C. The phosphoamino acids were separated by single dimensional thin layer chromatography on cellulose pre-coated plates using isobutyric acid and 0.5 N NH4OH (5:3 v/v) as a solvent system. Radiolabelled amino acids were detected by autoradiography and standard phosphoamino acids were visualized by staining with ninhydrin.
Other procedures SDS-PAGE was performed according to the method of Laemmli [12] on a 12% polyacrylamide gel with 4% stacking gel. Autoradiographs were made using KODAK-SAR-5 film. Protein was determined by the method of Lowry et al. [13] or Bradford [14] using BSA as standard.
We identified and purified Ca2+/CaM dependent protein kinase from C. albicans to apparent homogeneity. Initially the cytosolic fraction obtained after ultracentrifugation was subjected to 60% ammonium sulfate precipitation. The precipitate obtained after centrifugation was dialyzed. The protein precipitates after centrifugation and dialysis were subjected to DEAE-anion exchange chromatography. The protein was eluted using a linear NaCl gradient (0–600 mM) in buffer A. When calmodulin dependent protein kinase activity was assayed, enzyme activity emerged in three peaks (Fig. 1). In our earlier attempts, we tried to purify CaMPK using CaM agarose affinity column after DEAE-anion exchanger, but the eluted fractions resulted in 4–5 bands of CaM binding proteins on SDS-PAGE. So, before going to CaM agarose, we decided to first run a gel filtration column. The second major peak having maximum activity which eluted between 100–200 mM NaCl gradient of DEAE-ion exchange chromatography, was pooled, concentrated and loaded on to a Sephacryl S-300 gel permeation column. After gel filtration, one major broad peak, having CaM kinase activity was obtained (Fig. 2). Each fraction of this peak, when analyzed on SDS-PAGE followed by silver staining, showed a single band (Fig. 3). Figure 4 shows the protein profile of pooled active fractions at various purification steps. Twenty µg of protein aliquot from Sephacryl S-300 active fractions when loaded on 12% SDS-PAGE revealed a single band of 69 kDa. When this purified preparation was subjected to Superdex 75 gel filtration FPLC system, the enzyme eluted as a single pure peak at a position corresponding to 71 kDa (Figs 5a and 5b). Results of purification are summarized in Table 1. The specific activities of CaM kinase in crude extract and the purified preparation obtained were 5.48 and 3528 pmol/min/mg protein, respectively, indicating an overall purification of CaM kinase from the crude extract of about 644 fold. There was ~ 10 fold loss in the total activity between the homogenate and the sephacryl S-300 step and major loss occurred between application of ammonium sulphate precipitate protein to the DEAE column. This could be because of presence of two other CaMPK activity peaks (Fig. 2), which might contain other type of kinase. Activity of the major peak was compared with the total activity in the ammonium sulphate precipitate protein. Additionally, the loss could be due to dialysis. Effect of protein kinase inhibitors and calmodulin inhibitor on CaMPK activity To confirm that purified enzyme is a Ca2+/CaM dependent protein kinase, its phosphotransferase activity was deter-
186
Fig. 1. DEAE cellulose column chromatography using a linear NaCl gradient (0–600 mM).
Absorbance (280nm)
CaMPK activity
Fig. 2. Elution profile of Sephacryl S-300 gel permeation column chromatography.
187 Fr. No – 37
60
63
66
69
70
76
Fig. 3. SDS-PAGE profile of fractions from the Sephacryl S-300 gel filtration chromatography.
mined in the presence of different protein kinase inhibitors at different concentrations. W-7 (calmodulin inhibitor) and KN-62 (Ca2+/CaM protein kinase inhibitor) inhibited the activity of the enzyme in a dose dependent manner while PKI (specific PKA inhibitor) and H-7 (PKC and cyclic nucleotide dependent PK inhibitor) had no effect on protein kinase activity even when used at high concentrations, thus supporting the observation that purified enzyme is Ca2+/CaM dependent protein kinase (Fig. 6).
Autophosphorylation of purified enzyme When the purified enzyme was incubated with [γ-32P] ATP under protein phosphorylation conditions in the presence and absence of Ca2+/calmodulin and then analyzed by SDS-polyacrylamide gel electrophoresis; one radioactive band was observed in the presence of Ca2+/calmodulin, suggesting the
Fig. 5. Determination of the molecular mass of CaM kinase using Superdex G75. The peak fraction of CaM kinase activity after Sephacryl S-300 column was loaded on Superdex G75. (a) Elution profile of purified CaMPK from Superdex G75. (b) The gel filtration peak of CaM kinase corresponded to an apparent molecular mass of 71 kDa.
possibility of autophosphorylation of the enzyme (Fig. 7). Phosphoaminoacid analysis of autophosphorylated CaMPK showed phosphorylation of serine residues only (Fig. 8). Phosphorylation of tyrosine or threonine was not detected.
Kinetic properties Fig. 4. SDS-PAGE analysis of the purified Ca2+/CaM dependent protein kinase on a 12% gel by silver staining. (1) Molecular weight markers. Aliquot of pooled active fractions from (2) Sephacryl S-300 gel permeation column. (3) DEAE anionexchanger. (4) Aliquot after ammonium sulfate precipitation. (5) Cytosolic extract. (6) Crude.
It was of particular interest to determine the endogenous substrate specificity of purified CaM Kinase. The cytosolic extract of Candida whose calmodulin dependent protein kinase had
188 Table 1. Summary of the purification of Ca2+/CaM-dependent protein kinase from Candida albicans Purification step
Activity (pmol/min)
Protein (mg)
Sp. activity (pmol/min/mg prot)
Purification fold
% yield
Crude Cytosolic fract. Ammonium sulphate ppt. DEAE-cellulose Sephacryl S-300
20125 17980 15120 2366.3 2055
3670 2280 679.9 73.66 0.582
5.48 7.92 22.23 32.2 3528
1.0 1.5 4.1 5.9 643.8
100 89.34 75.13 11.75 10.21
been inactivated by heat treatment at 55°C was incubated with purified CaM kinase under phosphorylating conditions and incorporation of 32P into proteins was examined by SDSpolyacrylamide gel electrophoresis followed by autoradiography as shown in Fig. 9. CaM kinase phosphorylated many endogenous cytosolic proteins of 89, 84, 75, 59, 52, 49, 36, 33, 30 kDa thus, suggesting that purified enzyme may be involved in regulating different cellular functions. Phosphorylation of exogenous proteins and peptides also showed the broad substrate specificity. Syntide-2 (Pro-LeuAla-Arg-Thr-Leu-Ser-Val-Ala-Gly-Leu-Pro-Gly-Lys-Lys) and autocamtide-2 (Lys-Lys-Ala-Leu-Arg-Arg-Gln-Glu-ThrVal-Asp-Ala-Leu) were found to be best substrates followed by phosvitin, histone IIs and casein (Fig. 10). The kinetic properties of purified CaM kinase are compared with those of CaM kinase from Aspergillus and rat brain in Table 2. Km for syntide-2 and autocamtide-2 came out to be 20 and 62.5 µM respectively. Ka for calmodulin and Km for ATP were estimated to be 8.3 nM and 14.3 µM respectively. Both Ca2+ and Mg2+ were required for purified CaMPK to be fully active. Mn2+ ions also partially stimulated the kinase
activity whereas other divalent ions like Ni2+, Ba2+, Zn2+, Cu2+, Cd2+ had little or no effect. 5,5′-diothiobis-2-nitrobenzoic acid (DTNB) a sulfhydryl reagent (500 µM) inhibited the CaM kinase activity thus suggesting the involvement of sulfhydryl groups in the catalytic activity of the purified enzyme. CaMPK activity varied over a broad pH range with optimum activity at pH 8.0.
Fig. 6. Effect of various protein kinase inhibitors and calmodulin inhibitor on CaMPK enzyme activity. One hundred % activity corresponded to 73.69 pmols/min.
Fig. 7. Autophosphorylation of purified Ca2+/CaM dependent protein kinase from C. albicans in the presence (lane 1) and absence (lane 2) of Ca2+ and CaM.
Discussion We report here for the first time the purification and biochemical characterization of Ca2+/calmodulin dependent protein kinase from Candida albicans yeast cells. Purified protein on SDS-polyacrylamide gel electrophoresis corresponded to molecular weight of 69 kDa with an overall purification of 644 fold. The molecular weight of the native enzyme estimated from the gel filtration column was approximately 71 kDa thus indicating the monomeric structure of the purified CaMPK. Ca2+/CaM dependent protein kinases of 50–
189
Fig. 8. Autoradiography of phosphorylated amino acid(s) of phosphorylated crude extract (lane 1) and autophosphorylated CaMPK (lane 2). The positions of phosphoserine, phosphothreonine, phosphotyrosine are indicated by arrows.
70 kDa have earlier been reported in other yeasts like Saccharomyces cerevisiae [15, 16], Aspergillus nidulans [17] and Neurospora crassa [18].
Fig. 9. Endogenous protein phosphorylation of C. albicans by purified Ca2+/ CaM dependent protein kinase: Arrows indicate protein bands markedly phosphorylated by CaM kinase. CaM – calmodulin; PK – Ca2+/CaM dependent protein kinase; Ext – the crude extract of C. albicans.
Fig. 10. Protein substrate specificity of Ca2+/CaM dependent protein kinase of C. albicans. Substrate concentration 1 mg/ml except syntide-2, kemptide (40 µM) and autocamtide-2 (20 µM).
Autophosphorylation of protein kinases is common and is an obligatory step for full activation of some kinases. The purified protein of Candida exhibited autophosphorylation in the presence of Ca2+ and calmodulin like other reported CaM kinases [19–23]. Autophosphorylation of purified CaMPK in response to Ca2+ and calmodulin was at serine residues. This is similar to CaM kinase IV from rat cerebral cortex, in which Ser-437 was almost exclusively autophosphorylated [24]. There is need to be slow Ca2+/calmodulin dependent Ser 11 and Ser 12 autophosphorylation of CaM kinase IV that occurs in the absence of CaM kinase kinase [25]. Thus this enzyme can be classified as serine type protein kinase. CaM kinase inhibitor (KN-62) and calmodulin antagonist (W-7) inhibited the activity of the purified enzyme. On the other hand, CaMPK dependent protein kinase inhibitor and PKC inhibitor were unable to alter the activity of the purified enzyme, thus ensuring that the purified protein is CaM kinase. Previously Hidaka and Kobayashi [26] have reported that KN-62 competes with CaM and directly binds to CaM kinase. Phosphorylation of exogenous proteins and peptides showed the broad substrate specificity of purified CaM kinase like other eukaryotic kinases [27, 28]. Purified enzyme could also phosphorylate a large number of endogenous Candida proteins as seen by CaM kinase from rat cerebellum [29] and Ca2+/CaM protein kinase V from rat brain [30]. Thus, CaM kinase purified from C. albicans may be involved in the regulation of various cellular functions. Most of the divalent ions tested could not activate the enzyme except Mn2+ ions, which showed moderate activation. Fifty µM of Ca 2+ concentration was found to be optimum for phosphotransferase activity of purified CaMPK under the experimental conditions and concentrations of calmodulin higher than 200 nM inhibited the activity of purified CaM kinase
190 Table 2. Kinetic properties of purified CaM Kinase of C. albicans Parameter
CaMPK (Candida albicans)
Aspergillus CaMK [34]
Rat cerebellar CaMPK IV [42, 45]
Rat forebrain CaMK II [46]
Km for syntide-2 Km for autocamtide-2 Km for ATP Ka for calmodulin
20 µM 62.5 µM 14.3 µM 10 nM
– – 17 µM 55 nM
2.6–12 µM – 19–48 µM 32 nM
21.7 µM – 21.8 µM –
The apparent Km values for syntide-2, autocamtide-2 were obtained from double- reciprocal plots of data. The Km value for ATP and the Ka for calmodulin were measured with histone IIs as substrate. The Ka value for calmodulin was obtained from the concentration required for half maximal activation of the enzyme.
(data not shown). These results are in agreement with the studies of CaM kinases of rat brain [29–31]. It is suggested that CaM kinase II possesses two calmodulin binding sites, one with high affinity for calmodulin which is involved in the activation of the enzyme and a second, low affinity binding site, which is involved in the inactivation of the enzyme. Inhibition by thiol reagent indicates the presence of sulfhydryl group at or near the active site of CaM kinase of C. albicans as observed for Ca2+ dependent protein kinase of Paramecium tetraurelia [32]. Ca2+/CaM dependent protein kinase can transduce Ca2+ signal either by inducing modification of pre-existing proteins or by mediating cellular responses by activating molecules involved in the regulation of gene expression [33]. When commercially available CREB was used as a substrate in protein kinase assay, purified CaMPK was able to phosphorylate it (data not given), thus providing the evidence that purified kinase is a candidate for mediating the effect of Ca2+ on CREB activity. Further, work is in progress to identify and purify CREB like protein in Candida albicans and to elucidate the molecular mechanism of regulation of cell cycle and morphogenesis by Ca2+/CaM dependent phosphorylation in this yeast.
Acknowledgements This work was supported by grants from Council of Scientific and Industrial Research (CSIR), New Delhi, India.
References 1. 2.
3.
Manalan AS, Klee CB: Calmodulin. Adv Cyclic Nucleotide Prot Phosphoryl Res 18: 227–278, 1984 Goldenring JR, Goanzalez B, McGurie JS Jr, De Lorenzo RJ: Purification and characterization of calmodulin dependent kinase from rat brain cytosol able to phosphorylate tubulin and microtubulin associated protein. J Biol Chem 258: 12632–12640, 1983 Schulman H: The multifunctional Ca2+/calmodulin dependent protein kinase. Adv Second Messenger Phosphoprot Res 22: 39–112, 1988
4. Scherer S, Magee PT: Genetics of Candida albicans. Microbiol Rev 54: 226–241 1990 5. Wood, Nugent: Inhibitory effects of chlorpromazine on candida species. Antimicrob Agents Chemother 27: 692–694, 1985 6. Datta A, Ganesan K, Natarajan K: Current trends in Candida albicans research. Adv Microb Physiol 30: 53–88, 1989 7. Iida H, Sakaguchi S, Yagawa Y, Anraku Y: Cell cycle control by Ca2+ in Saccharomyces cerevisiae. J Biol Chem 265: 21216–21222, 1990 8. Lu KP, Means RA: Regulation of cell cycle by calcium and calmodulin. Endocrin Rev 14: 40–58, 1993 9. Sharma S, Kaur H, Khuller GK: Cell cycle effects of phenothiazines: Trifluoperazine and chlorpromazine in Candida albicans. FEMS Microbiol Lett 199: 185–190, 2001 10. Sharma S, Khuller GK: Changes in the cellular composition of C. albicans resistant to miconazole. Ind J Biochem Biophys 33: 420–424, 1996 11. Roskoski JR: Assays of protein kinases. Meth Enzymol 99: 3–6, 1983 12. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970 13. Lowry OH, Rosebrough NJ, Farr AL, Randal RJ: Protein measurement with folinphenol reagent. J Biol Chem 193: 265–275, 1951 14. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72: 248–256, 1976 15. Miyakawa T, Oka Y, Tsuchiya E, Fukui S: Saccharomyces cerevisiae protein kinase dependent on Ca2+ and calmodulin. J Bacteriol 171: 1417–1422, 1989 16. Londesborough J, Nuutinen M: Ca2+/calmodulin dependent protein kinase in Saccharomyces cerevisiae. FEBS Lett 219: 249–253, 1987 17. Bartelt DC, Fidel S, Farber LH, Wolff DJ, Hammell RL: Calmodulin dependent multifunctional protein kinase in Aspergilllus nidulans. Proc Natl Acad Sci USA 85: 3279–3283, 1988 18. Ulloa RM, Torres HN, Ochatt CM, Tellez-Inon MT: Ca 2+/calmodulin dependent protein kinase activity in the ascomycetes Neurospora crassa. Mol Cell Biochem 102: 155–163, 1991 19. Maeno H, Reyes PL, Veda T, Rudolph SA, Greengard P: Autophosphorylation of adenosine 3′,5′-monophosphate dependent protein kinase from bovine brain. Arch Biochem Biophys 164: 551–559, 1974 20. Rosen OM, Erlichman J: Reversible autophosphorylation of a cyclic 3′,5′-AMP dependent protein kinase from bovine cardiac muscle. J Biol Chem 250: 7786–7794, 1975 21. de Jonge HR, Rosen OM: Self phosphorylation of cyclic guanosine 3′,5′-monophosphate dependent protein kinase from bovine lung: Effect of cyclic adenosine 3′,5′-monophosphate, cyclic guanosine 3′,5′-monophosphate and kinase. J Biol Chem 252: 2780– 2783, 1977 22. Kikkawa V, Takai V, Minakuchi R, Inohara S, Nishizuka Y: Calcium activated, phospholipid dependent protein kinase from rat brain. J Biol Chem 257: 13341–13348, 1982
191 23. Hathaway DR, Adelstien RS: Human platelet myosin light chain kinase requires the calcium binding protein calmodulin for activity. Proc Natl Acad Sci USA 76: 1653–1657, 1979 24. Kameshita I, Fujisawa H: Autophosphorylation of calmodulin dependent protein kinase from rat cerebral cortex. J Biochem 113: 583–590, 1993 25. Chatila T, Anderson KA, Ho N, Means AR: A unique phosphorylation dependent mechanism for the activation of calcium/calmodulin dependent protein kinase type IV/GR. J Biol Chem 27: 21542–21548, 1996 26. Hidaka H, Kobayashi R: Pharmacology of protein kinase inhibitors. Annu Rev Pharmacol Toxicol 32: 377–392, 1992 27. Yuasa T, Muto S: Ca2+ dependent protein kinase form the halotolerant green alga Dunaliella tertiolecta: Partial purification and Ca2+ dependent association of the enzyme to the microsomes. Arch Biochem Biophys 296: 175–182, 1992
28. DeRiemer SA, Kaczmavek LK, Lai Y, McGuinness TL, Greengard P: Calcium/calmodulin dependent protein phosphorylation in the nervous system of Aplysia. J Neurosci 4: 1618–1625, 1984 29. Miyano O, Kameshita I, Fujisawa H: Purification and characterization of a brain specific multifunctional calmodulin dependent protein kinase from rat cerebellum. J Biol Chem 267: 1198–1203, 1992 30. Ito T, Mochizuki H, Kato M, Nimura Y, Hanai T, Usuda N, Hidaka H: Ca2+/calmodulin dependent protein kinase V: Tissue distribution and immunohistochemical localization in rat brain. Arch Biochem Biophys 312: 278–284, 1994 31. Ishida A, Kitani T, Okuno S, Fujisawa H: Inactivation of Ca2+/calmodulin protein kinase II by Ca2+/calmodulin. J Biochem 115: 1075– 1082, 1994 32. Gundersen RE, Nelson DL: A novel Ca2+/dependent protein kinase from Paramecium tetraurelia. J Biol Chem 262: 4602–4609, 1987
192