Higashi-Hiroshima, 724 Japan. Calmodulin (CaM) is a ubiquitous Ca2"-binding protein that regulates many cellular processes in eucaryotes.

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1 JOURNAL OF BACTERIOLOGY, Mar. 1989, P /89/ $02.00/0 Copyright 1989, American Society for Microbiology Vol. 171, No. 3 Saccharomyces cerevisiae Protein Kinase Dependent on Ca2' and Calmodulin TOKICHI MIYAKAWA,* YOUICHI OKA, EIKO TSUCHIYA, AND SAKUZO FUKUIt Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Saijo, Higashi-Hiroshima, 724 Japan Received 13 June 1988/Accepted 6 December 1988 A Ca2+- and calmodulin (CaM)-dependent protein kinase of Saccharomyces cerevisiae was partially purified by CaM affinity chromatography of the soluble fraction, and the properties of the enzyme were investigated. The protein kinase activity of the affinity-purified preparation was stimulated at least eightfold by the simultaneous presence of Ca2' and CaM. The enzyme stimulation was strongly inhibited by trifluoperazine (TFP), a CaM antagonist. When the kinase was incubated in the presence of ATP, Ca2+, and CaM before the assay, the enzyme showed activity even in the presence of the Ca2+ chelator ethylene glycol-bis(io-aminoethyl ether)-n,n,n',n'-tetraacetic acid (EGTA) and TFP. The conversion to this Ca2+- and CaM-independent form occurred very rapidly under the incubation conditions required for protein phosphorylation by the kinase. At the highest level of conversion, Ca2+- and CaM-independent kinase activity, which was measured in the presence of EGTA and TFP, was nearly equal to the total kinase activity, which was measured in the presence of Ca2' and CaM. A protein with a molecular weight of 58,000 was the major species that was phosphorylated in a Ca2+. and CaM-dependent manner by incubation of the CaM affinity-purified proteins with [y-32p]atp. The protein kinase activity of the protein with the same molecular weight was demonstrated by in situ protein phosphorylation in sodium dodecyl sulfate-polyacrylamide gels by using casein as the substrate, after removal of the detergent from electrophoresed CaM-binding proteins. These data indicate that phosphorylation of the kinase is responsible for the conversion of enzyme activity. Enzyme regulation by this mode may play an important role in integrating cellular functions during the cell cycle. A possible role for the Ca2+- and CaM-dependent protein kinase in the signal transduction of the mating pheromone a factor is also discussed. The mating process of the yeast Saccharomyces cerevisiae is regulated by mating pheromones (for a review, see reference 22). The haploid cell of the ao mating type produces a peptide mating pheromone a factor which arrests the cell division cycle of the haploid cell of the a mating type in the Gi phase of the cycle. Morphologically, the a factor elicits a localized elongation of the cell surface. These processes are assumed to be prerequisites for mating with the a cell, which undergoes similar changes by the action of another mating pheromone a factor produced by an a cell. The arrested a and a cells can then fuse to form a diploid cell. These cellular responses are thought to be mediated by the binding of the mating pheromones to specific receptors on the surface of the respective target cells. However, the signaling mechanism of the mating pheromones is not well understood. In the course of our studies on the mode of pheromone action in Rhodosporidium toruloides, a basidiomycetous yeast whose mode of sexual cell-cell interactions is analogous to that of S. cerevisiae (1), we found that a lipopeptidyl mating pheromone (rhodotorucine A) induces a very rapid and transient increase in the cellular Ca2+ level in the target cell (15, 16). Experiments with S. cerevisiae also showed similar characteristic Ca21 changes in mating type a cells in response to a factor (21). Thus, the primary cellular responses elicited by mating pheromones in yeasts appear to be an increase in cellular Ca2+ levels. * Corresponding author. t Present address: Department of Biotechnology, Faculty of Engineering, Fukuyama University, Higashi-Murayama, Fukuyama, Japan Calmodulin (CaM) is a ubiquitous Ca2"-binding protein that regulates many cellular processes in eucaryotes. CaM has been found in yeasts (2, 18). It has properties similar to those of higher eucaryotes. The action of many mammalian hormones and other extracellular signals has been shown to be mediated by an elevation of the intracellular free Ca2+ level. The finding of Ca2+- and CaM-dependent protein kinases in brain and other tissues suggests that the protein kinase, by responding to the Ca2+ signal, may be involved in the regulation and coordination of numerous cellular processes (for a review, see reference 17). In the sexual signaling of yeasts, Ca2+- and CaM-dependent protein kinases might be involved in a similar manner, responding to the Ca2+ signal produced by the mating pheromones. However, little is known about yeast Ca2+- and CaM-dependent protein kinases. In the present study we demonstrate, using CaM-binding proteins isolated by CaM affinity chromatography from the cytosol proteins of S. cerevisiae, the presence of a Ca2'- and CaM-dependent protein kinase whose activity was modulated to a Ca2+- and CaM-independent form by Ca2'- and CaM-dependent phosphorylation. We suggest that there is a possibility that the modulation of kinase activity by autophosphorylation may play an important role in integrating cellular responses to mating pheromone, which acts as a Ca2+-triggered molecular switch in the cytoplasm. MATERIALS AND METHODS Microorganisms and growth conditions. A haploid strain of S. cerevisiae, A364A (a adel ade2 ural his7 lys2 tyri gall) was obtained from the Yeast Genetic Stock Center and was used for enzyme preparation. The cells were grown at 28 C

2 1418 MIYAKAWA ET AL. in 5-liter flasks containing 1 liter of YPG medium (14) on a rotary shaker (GlO Gyrotory shaker; New Brunswick Scientific Co., Inc., Edison, N.J.) at 200 rpm. A protease-deficient mutant (pep4) of S. cerevisiae (strain 20B-12) (6) was obtained from Yeast Genetic Stock Center and was used for CaM preparation. Preparation of CaM-binding proteins. All of the following operations were performed at 4 C, unless indicated otherwise. Cells were harvested at the logarithmic phase of growth (3 x 107 cells per ml) by centrifugation at 6,000 x g for 10 min and were washed twice with 200 ml each of 20 mm Tris hydrochloride (phi 7.0) containing 1 mm ethylene glycol-bis(p-anminoethyl ether)- N,N,N',N'-tetraacetic acid (EGTA). The washed cells were treated with Zymolyase 100T (60 pg/ml) in 6 ml of buffer A (20 mm HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid]- KOH [ph 7.0], 10 mlm 2-mercaptoethanol) containing 1 mm each of MgCl2 and EGTA for 30 min at 28 C. After the cells were washed with buffer A, they were suspended in 6 ml of buffer A containing protease inhibitors (1 mm phenylmethylsulfonyl fluoride [PMSF] and 5 p.g each of leupeptin, pepstatin, and phosphoramidon per ml). The Zymolyasetreated cells were then disrupted by passage through a French press (20,000 lb/in2). The broken cell suspension was centrifuged for 30 min at 20,000 x g. The supernatant was taken and further centrifuged for 60 min at 100,000 x g. The resulting clear supernatant was then loaded onto a DEAEcellulose column (1 by 13 cm) that was equilibrated with buffer A containing 0.1 mm EGTA. The column was washed with 10 column volumes of the same buffer before it was eluted in a stepwise manner with buffer A containing the following, which were added in the indicated order: (i) 0.06 M NaCl and 0.1 mam EOTA; (ii) 0.08 M NaCl and 0.1 mm EGTA; (iii) 0.22 M NaCl and 0.1 mm EGTA. Endogenous CaM remained bound to the ion exchanger under the column conditions. The Ca2' concentration of each eluate was adjusted to 1 mm by the addition of 0.1 M CaCl2. The CaM-depleted proteins were then applied to an affinity column (0.5 by 10 cm) of CaM-Sepharose 4B, which was prepared with.bovine brain CaM as described previously (20) and which was equilibrated in buffer A containing 0.1 mm Ca2+. To elute CaM-binding proteins, the affinity column was washed successively with 50 ml each of buffer A containing 0.1 mm CaC12, buffer A containing 0.1 mm CaCl2 and 0.2 M NaCl, and finally with buffer A containing 1 mm EGTA and 0.2 M NaCT. The eluate was dialyzed in a dialysis tube against two changes of 2 liters each of 20 mm Tris hydrochloride (ph 7.0). The dialysate was concentrated to about a 1/10 volume by dehydration of the dialysis bag against a dry powder of polyethylene glycol The concentrated sample was used for determination of protein kinase activity. Protein kinase assay. Protein kinase activity was measured in a reaction mixture (100 R,l) containing 20 mm Tris hydrochloride (ph 7.0), 10 mm MgCl2, 1.0 mm dithiothreitol, 0.02 mm [y-32p]atp (2,uCi), and 0.1 mg each of K-casein and bovine serum albumin per ml. When required, 0.5 mm CaCI2, 1 mm EGTA, 5 pug of CaM per ml, or 50,uM trifluoperazine (TFP) were added. CaM purified from S. cerevisiae (see below) was used in the experiments. The reaction was initiated by the addition of labeled ATP to the mixture, which was prewarmed to 28 C. After incubation at 28 C (usually for 10 min), the mixture was quickly applied onto filter paper (diameter, 23 mm; 3 MM; Whatman, Inc., Clifton, N.J) and immediately immersed in 10% trichloroacetic acid at 1000C (10 ml per filter). The filters were then J. BACTERIOL. washed three times with ethanol (5 min for each rinse, 10 ml per filter). The radioactivity on the filter was determined by liquid scintillation counting. To determine the effect of enzyme preincubation on the kinase activity, CaM-binding proteins were prephosphorylated under various conditions that were similar to those for the kinase assay, except that phosphorylation was carried out with unlabeled ATP (0.02 mm) in the absence of the exogenous substrate (K-casein). After the incubation, the enzyme activities were measured as described above by adding 2 pci ATP. Total and Ca2`and CaM-independent protein kinase activities were measured in the presence of 0.5 mm CaCl2 and 5 jig of CaM per ml and of 1 mm EGTA and 50 FM TFP, respectively. Analysis of posphorylted p. CaM-binding proteins were phosphorylated with [y-32p]atp by incubation in the presence of CaCl2 and CaM as described above for the protein kinase assay, except that K-casein was omitted from the incubation mixture. Phosphorylated proteins were precipitated in a microfuge tube by the addition of 100 IlI of 10% trichloroacetic acid in the presence of 20 jlg of bovine serum albumin, which was used as a carrier protein. The precipitates were collected by centrifigation in a microfuge and rinsed three times with 0.5 ml of ethanol. The washed precipitates were then dissolved in sample buffer for electrophoresis and analyzed by sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis by using the system of Laemmli (7) in 10o gels. The radioactivity on the gel was detected by autoradiography, as described previously (13). Gels were calibrated by using human erythrocyte membrane proteins and bovine serum albumin as molecular weight markers, as described previously (13). Detection of protein kinase activity in SDS-polyacry1unide gels. SDS-polyacrylamide slab gels (10% acrylamide) were prepared as described above, except that casein was included in the gel mixture at a final concentration of 1 mg/ml. CaM-binding proteins were solubilized in SDS sample buffer (7) by heating them at 100 C for 1 min before they were subjected to gel electrophoresis. Following electrophoresis, the gel was washed with four changes of 100 ml each of 25 mm HEPES buffer (ph 7.4) with gentle shaking over a period of 6 h at room temperature to remove SDS. The gel was then equilibrated for 1 h in 20 ml of 25 mm HEPES-10 mm MgCI2-1 jig of bovine CaM per ml-either 1 mm Ca2" or 1 mm EGTA. Then, [y-32p]atp (8 jim, 40 jici) was added, and the mixture was incubated for 6 h at room temperature to phosphorylate the casein. After the incubation, excess ATP was removed by rinsing the gel with six changes of 100 ml each of 40 mm HEPES (ph 7.4) over a period of 12 h. The radioactivity on the gel was detected by autoradiography after the gel was stained with Coomassie brilliant blue. Purification of CaM. CaM was purified from bovine brain by hydrophobic affinity chromatography by the procedure described by Gopalakrishna and Anderson (5) and was used to prepare CaM-Sepharose affinity columns. For the Ca2+and CaM-dependent protein kinase assay, CaM purified from S. cerevisiae by the following procedure was used. The cells of a peptidase-deficient strain (pep4) that were grown and harvested as described above for the preparation of CaM-binding proteins were suspended in 200 ml of buffer containing 50 mm Tris hydrochloride (ph 7.5), 1 mm 2- mercaptoethanol, 2 mm EDTA, 1 mm PMSF, and 5 jig each of leupeptin and pepstatin per ml. The cells were disrupted by passing them through a French press at 20,000 lb/cm2. The suspension of disriipted cells was centrifuged at 11,000 x g for 30 min. The solution was heated in a boiling water bath for 5 min, and the resulting protein coagulation was

3 VOL. 171, 1989 YEAST Ca2+- AND CALMODULIN-DEPENDENT PROTEIN KINASE 1419 Sc 1 CaM (+TFP) FIG. 1. Ca2+- and CaM-dependent protein kinase of S. cerevisiae. K-Casein was phosphorylated for 10 min by CaM affinitypurified proteins under the conditions indicated below. Incorporation of radioactivity from [y-32p]atp (0.02 mm, 2,uCi) into trichloroacetic acid precipitates was determined by scintillation counting. The incubation mixtures contained the following: 3,ug of CaM per ml and 0.5 mm Ca2" (bar 1); 3 pg of CaM per ml (bar 2); 0.5 mm Ca2+ (bar 3); none (bar 4); 3 jig of CaM per ml, 0.5 mm Ca2+, and 50,uM TFP (bar 5). removed by centrifugation as described above to obtain a clear solution. CaM was purified from the heat-treated solution by hydrophobic affinity chromatography (5). After the purification procedures, the preparation gave a single band by SDS-polyacrylamide gel electrophoresis that changed its electrophoretic mobility in the presence and absence of Ca2+. The protein concentration was determined by the method of Lowry et al. (11) by using bovine serum albumin as the protein standard. Usually, 1.5 to 2.0 mg of CaM was obtained from a 10-liter culture. Materials. K-Casein, TFP, PMSF, and adenosine 5'-[P,B,yimido]triphosphate (AMP-PNP) were purchased from Sigma Chemical Co. (St. Louis, Mo.). Leupeptin and pepstatin were obtained from Peptide Institute, Inc. (Osaka, Japan). Zymolyase 100T was purchased from Seikagaku Kogyo Inc. (Tokyo, Japan). [y-32p]atp was obtained from ICN Radiochemicals Inc. (Irvine, Calif.). CNBr-activated Sepharose was from Pharmacia Fine Chemicals (Piscataway, N.J.). CaM-Sepharose 4B was prepared by coupling bovine brain CaM to CNBr-activated Sepharose 4B by the procedure recommended by the manufacturer. Usually, 1 mg of CaM was coupled per ml of gel. RESULTS Ca2+- and CaM-dependent protein kinase from S. cerevisiae. Since yeast cells contain many species of protein kinases, it was anticipated that detection of Ca2+- and CaM-dependent protein kinase activity by using the wholecell extract would be very difficult. Thus, CaM-binding proteins prepared by CaM affinity chromatography were used as the source of enzymes to measure the protein kinase activity. To remove CaM, the cell extract was first applied to a column of DEAE-cellulose in the presence of EGTA. The proteins that eluted from the DEAE-cellulose column with 0.08 M NaCl were further subjected to CaM affinity chromatography. By weight, the CaM-binding proteins made up approximately 0.013% of the total soluble proteins. Ca2+and CaM-dependent protein kinase activity was assayed with [-y-32p]atp as the phosphate donor and K-casein as the substrate (Fig. 1). The kinase activity was stimulated about eightfold by the simultaneous addition of Ca2" and CaM, showing the presence of Ca2+- and CaM-dependent protein kinase in the CaM-binding protein preparation. The presence of either Ca2" or CaM alone was not sufficient for enzyme activation. Ca2" had a significant effect on the enzyme activity. TFP, a CaM antagonist, strongly inhibited the kinase activity. A protein fraction obtained by further elution of the DEAE-cellulose column with 0.22 M NaCl, followed by CaM affinity chromatography, also showed considerable protein kinase activity (ca. 75% of the activity in the fraction eluted with 0.08 M NaCl). However, Ca2+ and CaM stimulated this kinase activity by only 10% (data not shown). The protein kinase activity in this fraction may represent phosphorylated species of the major Ca2+- and CaM-dependent protein kinase as described below. The Ca2'- and CaM-dependent protein kinase activity was not present at a detectable level in the CaM-binding proteins isolated from Nonidet P-40-solubilized membrane proteins (data not shown). Only the Ca2+- and CaM-dependent protein kinase of the soluble fraction derived from the DEAE-cellulose column eluted with 0.08 M NaCl. The CaM affinity column is further characterized below. Conversion to a Ca2+- and CaM-independent protein kinase. The effects of preincubating the Ca2+- and CaMdependent protein kinase with Ca2+ and CaM and ATP on the enzyme activity were determined (Fig. 2). CaM-binding proteins were preincubated under the complete conditions for phosphorylation (i.e., in the presence of Ca2, CaM, and unlabeled ATP) for various periods of time in the absence of K-casein, and then the Ca2'- and CaM-independent kinase activities (assayed in the presence of EGTA and TFP) and the total protein kinase (assayed in the presence of Ca2+ and CaM) were determined for each sample. A progressive increase in protein kinase activity that did not require exogenously added Ca2+ and CaM occurred with the time of preincubation. The change of the activity was very rapid and IC Total activity Ca2+/CaM_ independent >0.4 t activity U O lo Proincubation, min FIG. 2. Time course of generation of the Ca2+ and CaM-independent form of protein kinase on incubation of Ca2+ - and CaMdependent protein kinase in the presence'of Ca +, CaM, and ATP. After various periods of preincubation, protein kinase activity was assayed for 10 min in the presence of Ca2' and CaM for the total activity (O) or EGTA and TFP for the Ca2+- and CaM-independent activity (e), with K-casein used as the substrate.

4 1420 MIYAKAWA ET AL. J. BACTERIOL. MW A B 58I- _ I +AMP-PNP Preincubation, min FIG. 3. Effect of various preincubation conditions on the generation of Ca2+- and CaM-independent kinase activity from Ca2+- and CaM-dependent protein kinase. CaM-binding proteins were preincubated in a mixture containing the following: Ca2", CaM, and ATP (complete) (0); Ca2" and ATP (-CaM) (0); CaM and ATP, (-Ca2") (A); Ca2` and CaM (-ATP) (l); Ca2", CaM, and AMP- PNP (x). Preincubation was carried out for 0, 5, and 10 min. Protein kinase activity was assayed for 10 min in the presence of Ca2" and CaM for total kinase activity (dotted line) and in the presence of excess EGTA and TFP for Ca2+- and CaM-independent kinase activity (solid line). was completed within 5 min of preincubation (Fig. 2). The maximum Ca2+- and CaM-independent activity achieved by the preincubation was almost equivalent to the total kinase activity. Both activities declined with a prolonged preincubation period. The activity after 60 min of preincubation was about 50% of the control activity (data not shown). The requirements for enzyme conversion were investigated. When Ca2, CaM, or unlabeled ATP was omitted from the preincubation mixture, the conversion to the Ca2+and CaM-independent form did not occur (Fig. 3). The results of these experiments indicate that Ca2, CaM, and ATP are all essential for the generation of the Ca2+- and CaM-independent form of the enzyme, which is consistent with the requirements for Ca2+- and CaM-dependent protein kinase activity. The effect of the presence of the nonhydrolyzable ATP analog AMP-PNP during the preincubation period on the enzyme conversion was examined. No detectable extent of conversion to the Ca2+- and CaM-independent form occurred by incubating the enzyme in the presence of AMP-PNP (Fig. 4). In this experiment, the total kinase activity measured in the presence of AMP-PNP was about 50% of the control, possibly because of competitive inhibition of the protein kinase activity by the analog. (The concentration of the analog that was present during the enzyme assay was equal to that of ATP.) The results indicate that protein phosphorylation is involved in enzyme conversion. Phosphorylation of CaM-binding proteins. Protein molecules that were phosphorylated endogenously by incubating CaM-binding proteins with [iy-32p]atp were analyzed by SDS-polyacrylamide gel electrophoresis. When phosphorylation of the proteins was performed in the presence and Ca2'CaM - + FIG. 4. (A) Endogenous phosphorylation of CaM-binding proteins. CaM-binding proteins were labeled with [y-32p]atp in the presence and absence of Ca2" and CaM. The labeled samples were analyzed by SDS-polyacrylamide gel electrophoresis (10% gel). (B) Detection of protein kinase activity in an SDS-polyacrylamide gel. The protein kinase activity of CaM-binding proteins was localized by in situ protein phosphorylation by using the gel overlay method, following separation of the proteins by SDS-polyacrylamide gel electrophoresis (10%o acrylamide) and removal of SDS from the gel. The gels were incubated in the presence of Ca2" and CaM or 1 mm EGTA for phosphorylation. Molecular weights are indicated in thousands. and CaM, proteins with molecular weights of 58,000 and 71,000 were labeled under the two conditions (Fig. 4A). Among these, the 58,000-dalton protein was phosphorylated markedly in a Ca2+- and CaM-dependent manner. To identify the catalytic subunit of the protein kinases in the CaM affinity-purified protein fraction, we used the gel overlay method developed by Geahlen et al. (4). CaM-binding proteins were first separated by SDS-polyacrylamide gel electrophoresis in a gel containing 1 mg of casein per ml. SDS was removed from the electrophoresed proteins by extensive washing of the gel with buffer, and then phosphorylation was performed by incubating the gel with 40,uCi [.y-32p]atp in the presence or absence of Ca2" and CaM (Fig. 4B). A radioactive band labeled in a Caand CaM-dependent manner appeared at the position corresponding to a molecular weight of 58,000. The results indicate that the 58,000-dalton protein labeled by endogenous phosphorylation is itself a protein kinase. absence of Ca2" DISCUSSION In the present study, we demonstrated a Ca2+- and CaM-dependent protein kinase activity among the CaMbinding proteins of the cytosol fraction of S. cerevisiae. The enzyme activity was stimulated severalfold by the simultaneous presence of Ca2' and CaM (Fig. 1). When the enzyme was preincubated under the conditions for phosphorylation, the protein kinase was rapidly converted to a form that was active even in the absence of exogenously added stimulators (Fig. 2). Ca2+- and CaM-independent activity at the highest level of the enzyme conversion was nearly equal to the total kinase activity measured in the presence of Ca2+ and CaM. The enzyme conversion occurred only under conditions required for the phosphorylation of an exogenous substrate K-casein by the Ca2+- and CaM-dependent kinase (Fig. 3). A nonhydrolyzable ATP analog AMP-PNP could not substitute - +#

5 VOL. 171, 1989 YEAST Ca2+- AND CALMODULIN-DEPENDENT PROTEIN KINASE 1421 for ATP, suggesting that the transfer of phosphate is required for enzyme conversion. Protein bands with molecular weights of 58,000 and 71,000 that were labeled in a Ca2+- and CaM-dependent manner by endogenous phosphorylation of the CaM-binding proteins are likely to be candidates for Ca2+- and CaM-dependent protein kinase (Fig. 4A). The protein kinase activity of the 58,000-dalton protein was demonstrated by in situ protein phosphorylation in SDS-polyacrylamide gels after removal of SDS from electrophoresed CaM-binding proteins (Fig. 4B). These results suggest that phosphorylation of the 58,000-dalton protein is most likely responsible for the generation of Ca2+- and CaM-independent activity. To determine definitively that the modulation of yeast kinase activity is indeed caused by autophosphorylation, characterization of the purified enzyme is necessary. Little change in the total kinase activity, which was measured in the presence of Ca2" and CaM, was observed before or after the enzyme conversion (Fig. 3 and 4), indicating that the kinase is specifically affected in its Ca2' and CaM requirement. The Ca2+- and CaM-independent kinase activity decreased slowly to about 50% of the original activity after 60 min of preincubation (data not shown). It may have been caused by the thermal instability of phosphorylated enzyme (Ca2+- and CaM-independent kinase) in the in vitro reaction system that was used, as has been suggested to occur with mammalian enzymes (8). Alternatively, the inactivation may have a physiological role in the signaling system to eliminate the signal once it is generated. Little is known about yeast Ca2+- and CaM-dependent protein kinases. The presence of Ca2+- and CaM-dependent protein kinase(s) was recently reported in CaM affinitypurified soluble proteins of S. cerevisiae (9). The enzyme was not characterized, but from the procedures of the enzyme preparation, the protein kinase activity reported by the authors appears to be identical to ours. The yeast Ca2+and CaM-dependent protein kinase revealed in the present study has features very similar to those of mammalian Ca2+and CaM-dependent protein kinase (type II CaM kinase) whose activities have been reported to be regulated by autophosphorylation (8, 10, 12, 19). The autonomous enzyme modulation in brain tissue is assumed to play an important role in signal transduction. Ca2+ and CaM are indispensable for the growth of yeasts (3). The enzyme may play an important role in integrating cellular functions during the cell cycle. The yeast Ca2+- and CaM-dependent protein kinase may also be involved in the signal transduction of mating pheromones. It seems likely that the enzyme becomes phosphorylated in a Ca2+- and CaM-dependent manner because of a short rise in the intracellular free Ca2+ concentration induced by mating pheromone (21), and the modified enzyme remains active after the intracellular Ca2+ concentration returns to basal levels. Thus, the yeast Ca2+and CaM-dependent protein kinase could be an essential component of the signal transducing machinery of mating pheromone, functioning as a molecular switch that remains active after the decay of the transient Ca2+ signal. It will be important to determine whether protein kinase is independent of its activators in vivo. ACKNOWLEDGMENT This work was supported in part by grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan. LITERATURE CITED 1. Abe, K., I. Kusaka, and S. Fukui Morphological change in the early stage of the mating process of Rhodosporidium toruloides. J. Bacteriol. 122: Davis, T. N., and J. Thorner Calmodulin and other calcium-binding proteins in yeast. UCLA Symp. Mol. Cell. Biol. 33: Davis, T. N., M. S. Urdea, F. R. Masiarz, and J. Thorner Isolation of the yeast calmodulin gene: calmodulin is an essential protein. Cell 47: Geahlen, R. L., M. Anostario, Jr., P. S. Low, and M. L. Harrison Detection of protein kinase activity in sodium dodecyl sulfate-polyacrylamide gels. Anal. Biochem. 153: Gopalakrishna, R., and W. B. Anderson Ca2"-induced hydrophobic site on calmodulin: application for purification of calmodulin by phenyl-sepharose affinity chromatography. Biochem. Biophys. Res. Commun. 104: Hemmings, B. A., G. S. Zubenko, A. Hasilik, and E. W. Jones Mutant defective in processing of an enzyme located in the lysosome-like vacuole of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 78: Laemmli, U. K Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: Lai, Y., A. C. Nairn, and P. Greengard Autophosphorylation reversibly regulates the Ca2+/calmodulin-dependence of Ca2+/calmodulin-dependent protein kinase II. Proc. Natl. Acad. Sci. USA 83: Londesborough, J., and M. Nuutinen Ca2+/calmodulindependent protein kinase in Saccharomyces cerevisiae. FEBS Lett. 219: Lou, L. L., S. J. Lloyd, and H. Schulman Activation of the multifunctional Ca2+/calmodulin-dependent protein kinase by autophosphorylation: ATP modulates production of an autonomous enzyme. Proc. Natl. Acad. Sci. USA 83: Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Miller, S. G., and M. B. Kennedy Regulation of brain type II Ca2+/calmodulin-dependent protein kinase by autophosphorylation: a Ca2'-triggered molecular switch. Cell 44: Miyakawa, T., T. Kadota, Y. Okubo, T. Hatano, E. Tsuchiya, and S. Fukui Mating pheromone-induced alteration of cell surface proteins in the heterobasidiomycetous yeast Tremella mesenterica. J. Bacteriol. 158: Miyakawa, T., M. Nishihara, E. Tsuchiya, and S. Fukui Role of metabolism of mating pheromone in sexual differentiation of the heterobasidiomycete Rhodosporidium toruloides. J. Bacteriol. 151: Miyakawa, T., T. Tachikawa, R. Akada, E. Tsuchiya, and S. Fukui Involvement of Ca2+/calmodulin in sexual differentiation induced by mating pheromone rhodotorucine A in Rhodosporidium toruloides. J. Gen. Microbiol. 132: Miyakawa, T., T. Tachikawa, Y. K. Jeong, E. Tsuchiya, and S. Fukui Transient increase of Ca2+-uptake as a signal for mating-pheromone-induced differentiation in the heterobasidiomycetous yeast Rhodosporidium toruloides. J. Bacteriol. 162: Nairn, A. C., H. C. Hemmings, Jr., and P. Greengard Protein kinases in the brain. Annu. Rev. Biochem. 54: Ohya, Y., I. Uno, T. Ishikawa, and Y. Anraku Purification and biochemical properties of calmodulin from Saccharomyces cerevisiae. Eur. J. Biochem. 168: Schworer, C. M., R. J. Colbram, and T. R. Sorderling Reversible generation of a Ca2+ (calmodulin)-dependent protein kinase II by an autophosphorylation mechanism. J. Biol. Chem. 261: Sharma, R. K., W. A. Taylor, and J. H. Wang Use of

6 1422 MIYAKAWA ET AL. calmodulin affinity chromatography for purification of specific calmodulin-dependent enzymes. Methods Enzymol. 102: Tachikawa, T., T. Miyakawa, E. Tsuchiya, and S. Fukui A rapid and transient increase of cellular Ca2" in response to mating pheromone in Saccharomyces cerevisiae. Agric. Biol. J. BACTERIOL. Chem. 51: Thorner, J Pheromonal regulation of development in Saccharomyces cerevisiae, p In J. N. Strathern, E. W. Jones, and J. R. Broach (ed.), The molecular biology of the yeast Saccharomyces cerevisiae: life cycle and inheritance. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.