Phosphorylation of liver plasma membrane-bound

Phosphorylation of liver plasma membrane-bound calmodulin'

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SHOBHAGHOSH, JONG. CHURCH,BASIL D. ROUFOGALIS,AND ANTONIOVILLALOBO~ Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, B.C. Canada V6T 1W5 Received October 1, 1987 B. D., and VILLALOBO, A. 1988. Phosphorylation of liver plasma membrane-bound GHOSH,S., CHURCH,J. G., ROUFOGALIS, calmodulin. Biochem. Cell Biol. 66: 922-927. In highly purified rat liver plasma membrane preparations, membrane-bound calmodulin was phosphorylated by a membrane-bound protein kinase using [ y - 3 2 ~as]phosphate ~ ~ ~ donor. Maximum phosphorylation of calmodulin occurred in the absence of calcium ion, but was significantly decreased in its presence. Plasma membrane-bound calmodulin was identified by the following criteria: (i) extraction from the membrane by EGTA, (ii) stimulation of the activity of the ca2+-calmodulindependent enzyme, (3':S1AMP)-phosphodiesterase,by the EGTA extract, and (iii) electrophoretic comigration of EGTAextracted protein with standard bovine brain calmodulin, both in the presence and the absence of Ca2+. Phosphorylation of the plasma membrane-bound calmodulin was shown by electrophoretic comigration of the 32~-labelled molecule with bovine brain calmodulin, the absence of phosphorylation of this protein band in calmodulin-depleted membranes, and a Western blot of the phosphorylated band using a calmodulin antibody. Treatment of plasma membrane preparations with sheep anticalmodulin serum prevented the phosphorylation of the calmodulin band. Phosphocalmodulin, which could be partially extracted from the membrane by EGTA, comigrated with bovine brain calmodulin in polyacrylamide gel electrophoresis. B. D., et VILLALOBO, A. 1988. Phosphorylation of liver plasma membrane-bound GHOSH,S., CHURCH,J. G., ROUFOGALIS, calmodulin. Biochem. Cell Biol. 66 : 922-927. Dans des prkparations hautement purifikes de membranes plasmiques de foie de rat, la calmoduline lite i la membrane est phosphorylke par une protkine kinase membranaire avec le [ y - 3 2 ~comme ] ~ ~donneur ~ de phosphate. La phosphorylation de la calmoduline est maximale en absence de I'ion calcium et elle est diminute de fagon significative en sa prtsence. Les critkres suivants permettent d'identifier la calmoduline like A la membrane plasmique: (i) extraction de la membrane par I'EGTA, (ii) stimulation de I'activitk de la (3':5'AMP)-phosphodiestkrase, enzyme dkpendante de la Ca2+-calrnoduline,par l'extrait obtenu avec I'EGTA et (iii) comigration klectrophorktique de la protkine extraite par I'EGTA avec la calmoduline standard de cerveau de boeuf en prksence et absence de Ca2+.La phosphorylation de la calmoduline like i la membrane plasmique est dkmontr6e par la comigration klectrophorktique de la molkcule marqute au 3 2 avec ~ la calmoduline de cerveau de boeuf, par I'absence de phosphorylation de cette bande protkique dans les membranes dkmunies de calmoduline et par buvardage Western de la bande phosphorylke i I'aide d'un anticorps anticalmoduline. Le traitement des pdparations de membranes plasmiques avec un strum de mouton anticalmoduline em@che la phosphorylation de la bande calmoduline. La phosphocalmoduline, qui peut 2tre partiellement extraite de la membrane par I'EGTA, se dkplace en mCme temps que la calmoduline de cerveau de boeufs dans I'klectrophor~sesur gel de polyacrylamide. [Traduit par la revue]

Introduction

ed (Jackson et al. 1977; Gagnon e t al. 1981), although the physiological role of this covalent modification in the regulation of its functions remains controversial (Gagnon et al. 1981; Billingsley e t al. 1983, 1984; Bmnauer and Clarke 1986). Recently, phosphorylation of calmodulin has also been reported in different tissues and implicated in several physiological functions (Plancke and Lazarides 1983; Lin e t al. 1986; Graves et al. 1986; Nakajo et al. 1986; Fukami e t al. 1986; Meggio e t al. 1987; Colca et al. 1987). T h e present paper demonstrates for the first time the phosphorylation of plasma membrane-bound calmodulin by a plasma membraneABBREVIATIONS: FITC, fluorescein isothiocyanate; M,, bound protein kinase in purified plasma membrane relative mass. preparations from rat liver and its isolation b y EGTA 'Supported by grants 2(86-1) and 4(87-1) from the British treatment of the membrane. Columbia Health Care Research Foundation, and grants 84-0247 and 85-1020 from the Cancer Research Society Inc. Materials and methods Montreal, Que. to A.V. Chemicals 2 ~ u t h otor whom correspondence and reprint requests are to [ y - 3 2 ~ (tetratriethylamrnonium ] ~ ~ ~ salt) (28.9 Ci.mbe sent. mol-', 1 Ci = 37 GBq) was purchased from ICN (Toronto, Regulation of a large number of ca2+-dependent cellular functions is mediated by calmodulin (Klee et al. 1980; Means and Dedman 1980; Klee and Vanaman 1982; Veigl e t al. 1984; Manalan and Klee 1984). Although the levels of intracellular concentrations of Ca2+ appear to control the activation and deactivation of the different Ca2+-calmodulin systems, the posttranslational modification of the calmodulin molecule in a reversible manner could further regulate those cellular functions. Methylation of calmodulin has been describ-

NOTES

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Ont.), and X-OMAT AR X-ray blue sensitive film was from Kodak. Bovine brain (3':S1AMP)-phosphodiesterase (activator-deficient), 5 '-nucleotidase from Crotalus adamanteus venom, and FITC-labelled protein A from Staphylococcus aureus were obtained from Sigma Chemical Co. (St. Louis, MO). Bovine brain calmodulin was purchased from Calbiochem (La Jolla, CA) or Sigma Chemical Co., and the sheep anticalmodulin serum was obtained from Diversified Biotech Inc. (Newton Centre, MA). All other chemicals used in this work were of analytical grade. Preparation of plasma membrane fractions Plasma membrane fractions from adult rat liver were prepared as described previously (Church et al. 1988). When calmodulin-depleted plasma membranes were required, the isolation was performed with 1 mM EGTA in all buffers and sucrose gradients. EGTA was removed in the final centrifugation by washing the membranes in EGTA-free medium. With respect to crude homogenate, purified plasma membranes were enriched 25-fold and 60-fold in ouabain-sensitive Na+, K+-ATPase and 5'-nucleotidase, respectively. Plasma membrane phosphorylation and extraction of phosphocalmodulin Plasma membrane phosphorylation was carried out as describedpreviously (Church et al. 1988) using [y-32~]ATP as phosphate donor. For the extraction of phosphocalmodulin, larger quantities of membranes were phosphorylated in the presence of 0.5 mM EGTA and 0.5 mM sodium vanadate to prevent dephosphorylation (Church et al. 1988). After 1 min, EGTA was added to a final concentration of 2 rnM and incubation continued for 10 min. The reaction mixture was thereafter centrifuged at 56000 x g,, for 30 min. The supernatant was treated with 10% (w/v) trichloroacetic acid to precipitate the EGTA-extracted proteins. The precipitate was collected by centrifugation at 3000 x g, for 30 min and processed for electrophoresis. Membrane-bound calmodulin was extracted by incubating plasma membranes with 2 m M EGTA at 37°C for 10 min or, alternatively, at 100°C for 10 for 30 min, the min. After centrifugation at 56 000 X g,, supernatant was dialyzed for 48 h against 10 x 1 L of distilled water and concentrated by lyophilization. The lyophilized powder was then used for electrophoresis or phosphodiesterase assays. Analytical procedures Slab gel electrophoresis (Laemmli 1970) was performed at 12 rnA for 16 h in a linear gradient of 5 to 20% (w/v) polyacrylmide gel in the presence of 0.1% (w/v) sodium dodecyl sulfate (pH 8.3). When required, 10 p,M CaC12 was added during gel polymerization and in the running buffer. The gels were stained with Coomassie Brilliant Blue R-250, and after drying the gel under vacuum on top of Whatman no. 1 filter paper, an X-ray film was exposed at -70°C for 48-72 h. For Western blot (Jarausch and Kadenbach 1982), proteins were electrophoretically transferred from the gel onto nitrocellulose paper and incubated with anticalmodulin serum. The calmodulin-I~G complex was visualized by UV light after incubation of calmodulin with ~roteinA labelled with the fluorescent probe FITC and removal of excess FlTC - protein A. Phosphodiesterase activity was assayed by coupling the

FIG. 1. Identification of plasma membrane-bound phosphocalmodulin. Track 1. Standard protein markers: myosin (MI 200 OOO), P-galactosidase (MI 116 250), phosphorylase B (M, 92 500), bovine serum albumin (MI 66 200), ovalbumin (M, 45 OOO), carbonic anhydrase (MI 31 OOO), soybean trypsin inhibitor (MI 21 500), lysozyme (MI 14400). Track 2. Coomassie Blue staining of 5 p,g bovine brain calmodulin. Track 3. Negative print of the fluorescence of the FITC protein A - Immunoglobulin G - calmodulin complex under UV light. Western blots was performed as described in Materials and methods. The results are representative of three different experiments, in duplicate, using plasma membranes preincubated with [ y - 3 2 ~ ] ~The T ~fluorescent . band perfectly matched the 16.5-KDa phosphoprotein band (not shown). hydrolysis of 3'3'-AMP to 5'-nucleotidase and determining the inorganic phosphate liberated from AMP by a colorimetric method (Raess and Vincenzi 1980). Protein concentration was determined by the method of Lowry et al. (1951), after precipitating the protein with 10% (w/v) trichloroactic acid and using bovine serum albumin as standard. Calmodulin concentration in the EGTA extract was deter-

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FIG.2. Effect of sheep anticalmodulin serum on calmodulin phosphorylation. Left. Plasma membranes (1 16 pg protein) suspended in a 100-pL aliquot of 10 mM MgC12, 25 mM Na-Hepes (pH 7.4) were incubated at 4OC for 30 min in 50 FL 0.9% (w/v) NaCl, 1 mM phosphate buffer (pH 7.4) (C sample) or 50 pL sheep anticalmodulin serum (AS sample). The two samples were incubated thereafter for 1 rnin with 5 p M [ y - 3 2 ~(10 ] pCi). ~ ~ ~After exposure to [ y - 3 2 ~was ] ~completed, ~ ~ the control plasma membranes (C) were also treated with the same amount of antiserum as their AS counterpart. Both samples (30 pg plasma membrane protein) were processed for electrophoresis and autoradiography as described in Materials and methods. A plot of the photodensitometric scan of the X-ray film at 700 nm is presented. The position of phosphocalmodulin (16.5 KDa) is indicated by an arrow. Right. The experiment was performed as above, except that the preincubation of the plasma membrane with the antiserum was carried out at 20°C. Arrow heads indicate the position of phosphocalmodulin (16.5 KDa). The results present two different experiments done in duplicate. mined by densitometric scanning of the Coomassie Bluestained gels and integration of the area of the peak with a planimetre. Free calcium concentration was calculated by a modified computer program (CATIONS, BC) described by Goldstein (1979).

for IgG (Langone 1978). Comigration of the 16.5-KDa pho~phoprotein(not shown) with a 16.5-KDa fluorescent band was observed in the nitrocellulose paper under a uv lamp (Fig. 1, track 3); this band comigrated also with standard bovine brain calmodulin (Fig. 1, tract 2).

Results IdentiJication of plasma membrane-bound phosphocalmodulin To further identify the nature of the 16.5-KDa phosphoprotein described by us (Church et al. 1988) and present in purified liver plasma membrane, we performed a Western blot of the 32~-labelled proteins using sheep anticalmodulin serum, and we incubated the phosphoprotein with a fluorescent FITC-labelled protein A from Staphylococcus aureus, known to have affinity

Inhibition of calmodulin phosphorylation by anticalmodulin serum To further ascertain the nature of this 16.5-KDa phosphoprotein, we incubated the isolated plasma membranes with the sheep anticalmodulin serum and then with [ y - 3 2 ~ ]Figure ~ ~ ~2 .shows two different experiments with similar results; in the anticalmodulin serumtreated membrane (Fig. 2, trace and track AS), the 16.5-KDa phosphoprotein is virtually absent (arrow), in contrast with the control membrane in which the

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FIG. 3. Extraction of phosphocalmodulin by EGTA. Plasma membrane (1 10 pg protein) was incubated for 10 min at 37°C in 1.5 mL of 17.5 mM Na-Hepes (pH 7.4), 7 mM MgC12, 2 mM EGTA, 0.5 mM sodium vanadate, and 5 pM [ Y - ~ ~ P I A T (1P00 pCi). The reaction mixture was cooled and centrifuged at 56 000 x ,g for 30 min, and the supernatant was treated with ice-cold for 30 min. The acid-precipitated pellet was processed for 10% (w/v) trichloroacetic acid and centrifuged at 3000 X g, electrophoresis and the gel stained with Coomassie Blue (A). Autoradiography of the same preparation was performed as described in Materials and methods and scanned in a photodensitometer at 700 nm (B). Arrows indicate the position of phosphocalmodulin. The results are representative of five different experiments.

antiserum was added after the treatment with [y3 2 P ] A (Fig. ~ ~ 2, trace and track C). However, 3 2 ~ labelling of other polypeptides in both antiserum-treated and nontreated plasma membranes are identical in pattern and only sightly decreased in intensity.

Extraction of phosphocalmodulin from the plasma membrane Calmodulin was extracted from the plasma membranes by incubation with EGTA. After centrifugation of the sample, the clear supernatant was extensively dialyzed to remove EGTA and later lyophylized for concentration. The extracted material stimulated the activity of a purified bovine brain (3':5'AMP)phosphodiesterase preparation in a concentrationdependent and a Ca2+-dependent manner (results not shown). Extraction of the phosphorylated form of calmodulin was performed by a similar procedure after previous incubation of the membranes with [y-32P]ATP in the presence of vanadate. To minimize dephosphorylation of the 32~-labelledcalmodulin, however, the EGTA-

extracted proteins were precipitated with ice-cold trichloroacetic acid and processed for electrophoresis and autoradiography.Figure 3 (panel A) shows the Coomasie Blue-stained proteins from the EGTA extract, where the arrow points to the band of extracted phosphocalmodulin. Panel B of the same figure shows a densitometric scan of an autoradiogram of the same preparation, where the arrow points to the 32P-labelledphosphocalmodulin. In addition, other 32P-labelledphosphoproteins of higher molecular weight were present in the EGTA extract.

Discussion Earlier reports on the involvement of low molecular weight phosphoproteins (Wolff and Siegel 1972;Brooks and Siegel 1973a, 1973b) in the activation of calmodulin-dependent enzymes (Wolff and Brostrom 1974; Brostrom et al. 1975) were questioned when the subsequently purified activator was shown to lack covalently bound phosphate (see Cheung et al. 1975). Most recently, however, phosphorylation of calmodulin has been shown unequivocally to take place in several

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normal (Plancke and Lazarides 1983; Graves et al. 1986; Nakajo et al. 1986; Fukami et al. 1986; Meggio et al. 1987; Colca et al. 1987) and neoplastic (Lin et al. 1986; Fukami et al. 1986) tissues or cells. This report shows that plasma membrane-bound calmodulin in rat liver is phosphorylated by a plasma membrane-bound protein kinase, and this phosphorylated calmodulin can be extracted from the membranes, at least in part, by EGTA treatment. Calmodulin phosphorylation could take place in a variety of amino acid residues. Therefore, the apparently conflicting evidence of changes in the phosphocalmodulin electrophoretic mobility in the presence versus the absence of calcium, reported in some systems (Fukami et al. 1986) and absent in others (Plancke and Lazarides 1983), might be explained by phosphorylation in different amino acid residues: those essential to a conformational change induced by c a 2 + binding those in regions not involved in calcium binding. We have demonstrated that the electrophoretic mobility of the liver plasma membrane-bound phosphocalmodulin does not vary in the presence versus the absence of Ca2+ (results not shown), in contrast with the behaviour of the nonphosphorylated molecule (Burgess et al. 1980). The phosphorylation of rat liver plasma membranebound calmodulin is significantly inhibited by Ca2+, in agreement with a similar observaion of calmodulin phosphorylation by the purified src kinase in vitro (Fukami et al. 1986) and in contrast with the observation in A431 transformed cells (Lin et al. 1986). We have previously demonstrated that the ca2+-dependent inhibition of calmodulin phosphorylation is even more pronounced in neonatal rat liver and normal regenerating rat liver (Villalobo et al. 1986, 1987; Church et al. 1988). Since Ca2+controls many calmodulin-dependent functions, the modulation of calmodulin phosphorylation by Ca2+ could be a feedback mechanism of physiological importance. The physiological role of phosphocalmodulin in the liver plasma membrane is at present unknown. However, phosphocalmodulin is implicated in physiologically relevant systems such as the phosphorylase kinase system (Plancke and Lazarides 1983). Moreover, calmodulin phosphorylation has been shown most recently to be under the control of both insulin (Graves et al. 1986; Colca et al. 1987) and epidermal growth factor (Lin et al. 1986), and catalysed by the oncogene product src kinase in vitro (Fukami et al. 1986). The obvious implication is that phosphorylation of calmodulin could be a means to selectively modlfy the activities of various calmodulin-dependent systems. The 32P-labelled calmodulin recovered by EGTA extraction was only a fraction of the phosphocalmodulin bound to the plasma membrane. This could be either due to dephosphorylation of the molecule during the extrac-

tion procedure or result from an increased affinity of phosphocalmodulin for the plasma membrane. The extracted calmodulin was therefore a mixture of phosphorylated and nonphosphorylated molecues. Work in our laboratory is under way to separate the phosphorylated and nonphosphorylated forms of calmodulin to test whether or not the activation capacity of the molecule is affected by phosphorylation in a variety of calmodulindependent systems, both dependent and independent of calcium ion.

Acknowledgements We would like to thank Mr. Bruce Wilson for assisting us in the purification of the plasma membrane fractions, and Dr. Dorothy Jeffery for the calculation of the free calcium ion concentrations. BILLINGSLEY, M. L., VELLETRI, P. A . , ROTH,R. H . , and DELORENZO, R. J. 1983. Carboxylmethylation of calmodulin inhibits calmodulin-dependent phosphorylation in rat brain membranes and cytosol. J. Biol. Chem. 258: 5352-5357. BILLINGSLEY, M., KUHN,D., VELLETRI, P. A., KINCAID, R., and LOVENBERG, W. 1984. Carboxylmethylation of phosphodiesterase attenuates its activation by caz+-calmodulin. J. Biol. Chem. 259: 6630-6635. BROOKS, J. C., and SIEGEL,F. L. 1973a: Calcium-binding phosphoprotein: The principal acidic protein of mammalian sperm. Biochem. Biophys. Res. Cornmun. 55: 710-716. 1973b: Purification of a calcium-binding phosphoprotein form beef adrenal medules. Identity with one of two calcium-binding proteins of brain. J. Biol. Chem. 248: 4189-4193. BROSTROM, C. O., HUANG, Y.-C., BRECKENRIDGE, B. McL., and WOLFF,D. J. 1975. Identification of a calcium-binding protein as a calcium-dependent regulator of brain adenylate cyclase. Roc. Natl. Acad. Sci. U.S.A. 72: 64-68. BRUNAUER, L. S . , and CLARKE, S. 1986. Methylation of calmodulin at carboxylic acid residues in erythrocytes. A non-regulatory covalent modification? Biochem. J. 236: 811-820. BURGESS, W. H., JEMIOLO, D. K., and KRETSINGER, R. H. 1980. Interaction of calcium and calmodulin in the presence of sodium dodecyl sulfate. Biochim. Biophys. Acta, 623: 257-270. CHEUNG, W. Y., BRADHAN, L. S., LYNCH, T. J., LIN,Y. M., and TALLANT, E. A. 1975. Protein activator of cyclic 3'5'-nucleotide phosphodiesterase of bovine or rat brain also activates its adenylate cyclase. Biochem. Biophys. Res. Cornmun. 66: 1055- 1062. CHURCH,G. J., GHOSH,S., ROUFOGALIS, B. D., and VILLALOBO, A. 1988. Endogenous hyperphosphorylation in plasma membrane from an ascites hepatocarcinoma cell line. Biochem. Cell Biol. 66: 1-12. COLCA, J. R., DEWALD, D. B., PEARSON, J. D., PALAZUK, B. J., LAURINO, J. P., and MCDONALD, J. M. 1987. Insulin stimulates the phosphorylation of calmodulin in intact adipocytes. J. Biol. Chem. 262: 11 399 - 11 402. FUKAMI,Y., NAKAMURA, T., NAKAYAMA, A., and KANEH-

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ISA, T. 1986. Phosphorylation of tyrosine residues of RANDALL, R. J. 1951. Protein measurement with the Folin calmodulin in Rous sarcoma virus-transformed cells. Proc. phenol reagent. J. Biol. Chem. 193: 265-275. Natl. Acad. Sci. U.S.A. 83: 4190-4193. MANALAN, A. S., and KLEE,C. B. 1984. Calmodulin. Adv. GAGNON,C., KELLY,S., MANGANIELLO, V., VAUGHAN, Cyclic Nucleotide Protein Phosphorylation Res. 18: W., HOFFMAN, A., and M., ODYA,C., STRITTMATTER, 227-277. HIRATA, F. 1981. Modification of calmodulin function by MEANS,A. R., and DEDMAN, J. R. 1980. Calmodulin-an intracellular calcium receptor. Nature (London), 285: enzymatic carboxyl methylation. Nature (London), 291: 515-516. 73-77. GOLDSTEIN, D. A. 1979. Calculation of the concentrations of MEGGIO,F., BRUNATI,A. M., and PINNA,L. A. 1987. free cations and cation-ligand complexes in solutions Polycation-dependent, ca2+-antagonized phosphorylation containing multiple divalent cations and ligands. Biophys. of calmodulin by casein kinase-2 and a spleen tyrosine J. 26: 235-242. protein kinase. FEBS Lett. 215: 241-246. J. P., and MCDON- NAKAJO, GRAVES, C. B., GALE,R. D., LAURINO, S., HAYASHI, K., DAIMATSU, T., TANAKA, K., and ALD,J. M. 1986. The insulin receptor and calmodulin. Y. 1986. Phosphorylation of rat brain calmodNAKAMURA, Calmodulin enhance insulin-mediatedreceptor kinase activulin in vivo and in vitro. Biochem. Int. 13: 687-693. ity and insulin stimulates phosphorylation of calmodulin. J. PLANCKE, Y. D., and LAZARIDES, E. 1983. Evidence for a Biol. Chem. 261: 10429 - 10438. phosphorylated form of calmodulin in chicken brain and JACKSON, R. L., DEDMAN,J. R., SCHREIBER,W. E., muscle. Mol. Cell. Biol. 3: 1412-1420. BHATNAGAR, P. K., KNAPP,R. D., and MEANS,A. R. RAESS,B. U., and VINCENZI, F. F. 1980. A semi-automated 1977. Identification of E-N-trimethyllysine in a rat testis method for the determinationof multiple membrane ATPase calcium-dependent regulatory protein of cyclic nucleotide activities. J. Pharmacol. Methods, 4: 273-283. phosphodiesterase. Biochem. Biophys. Res. Commun. 77: VEIGL,M. L., VANAMAN, T. C., and SEDWICK, W. D. 1984. 723-729. Calcium and calmodulin in cell growth and transformation. JARAUSCH, J., and KADENBACH, B. 1982. Tissue-specificity Biochim. Biophys. Acta, 738: 21-48. ovemdes species-specificity in cytoplasmic cytochrome c VILLALOBO, A., CHURCH,J. G., and ROUFOGALIS, B. C. oxidase polypeptides. Hoppe-Seyler's Z. Physiol. Chem. 1986. Putative calmodulin phosphorylation in rat liver 363: 1133-1 140. plasma membrane and its absence in a hepatocarcinoma cell KLEE,C. B., and VANAMAN, T. C. 1982. Calmodulin. Adv. line. Proc. Int. Union Physiol. Sci. 16: 127. Protein Chem. 35: 213-321. VILLALOBO,A., CHURCH,J. G., ROUFOGALIS, B. D., KLEE,C. B., CROUCH,T. H., and RICHMAN,P. G. 1980. ROWAT,K. A., and LAU, N. 1987. Phosphorylation of Calmodulin. AMU. Rev. Biochem. 49: 489-515. plasma membrane-bound calmodulin. VII International Washington Spring Symposium Cell Calcium Metabolism LAEMMLI, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature '87. Abstr. no. 31. Washington, DC, May, 1987. (London), 227: 680-685. WOLFF,D. J., and BROSTROM, C. 0 . 1974. Calcium-binding LANGONE, J. J. 1978. ['z~]proteinA: A tracer for general use phosphoprotein from pig brain: Identification as a calciumin immunoassay. J. Immunol. Methods, 24: 269-285. dependent regulator of brain cyclic nucleotide phosphodiesLIN, P. H., SELINFREUND, R., and WHARTON, W. 1986. terase. Arch. Biochem. Biophys. 163: 349-358. Epidermal growth factor (EGF) sensitive phosphorylation WOLFF,D. J., and SIEGEL,F. L. 1972. Purification of a of calmodulin (CaM) in A431 cell membrane. Fed. Proc. calcium-binding phosphoprotein form pig brain. J. Biol. Fed. Am. Soc. Exp. Biol. 45: 1693. Chem. 247: 4180-4185. LOWRY,0 . H., ROSEBROUGH, N. J., FARR,A. L., and