Regulation of Calcineurin by Phosphorylation · Regulation of Calcineurin by Phosphorylation 16525 stream of N,, suspended in 200 p1 of HzO, and sonicated with a microtip sonicator

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 28, Issue of October 5, pp. 16524-16529,1989 Printed in U.S.A.

Regulation of Calcineurin by Phosphorylation IDENTIFICATION OF THE REGULATORY SITE PHOSPHORYLATED BY Ca2+/CALMODULIN-DEPENDENT PROTEIN KINASE I1 AND PROTEIN KINASE C*

(Received for publication, May 2, 1989)

Yoshiaki HashimotoS and Thomas R. SoderlingQ From the Howard Hughes Medical Institute and the Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, Tennessee 37232-0615

The site in calcineurin, the Ca2+/calmodulin (CaM)- dependent protein phosphatase, which is phosphorylated by Ca2+/CaM-dependent protein kinase I1 (CaM- kinase 11) has been identified. Analyses of "P release from tryptic and cyanogen bromide peptides derived from [32P]calcineurin plus direct sequence determination established the site as -Arg-Val-Phe-Ser(PO& Val-Leu-Arg-, which conformed to the consensus phosphorylation sequence for CaM-kinase I1 (Arg-X-X-Ser/ Thr-). This phosphorylation site is located at the C- terminal boundary of the putative CaM-binding domain in calcineurin (Kincaid, R. L., Nightingale, M. S. , and Martin, B. M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 898343987), thereby accounting for the observed inhibition of this phosphorylation when Ca2+/ CaM is bound to calcineurin. Since the phosphorylation site sequence also contains elements of the specificity determinants for Ca2+/phospholipid-dependent protein kinase (protein kinase C) (basic residues both N-terminal and C-terminal to Ser/Thr), we tested calcineurin as a substrate for protein kinase C. Protein kinase C catalyzed rapid stoichiometric phosphorylation, and the characteristics of the reaction were the same as with CaM-kinase 11: 1) the phosphorylation was blocked by binding of Ca2+/CaM to calcineurin; 2) phosphorylation partially inactivated calcineurin by in- creasing the K,,, (from 9.9 f 1.1 to 17.5 2 1.1 PM "P- labeled myosin light chain); and 3) [32P]calcineurin exhibited very slow autodephosphorylation but was rapidly dephosphorylated by protein phosphatase IIA. Tryptic and thermolytic 32P-peptide mapping and sequential phosphoamino acid sequence analysis confirmed that protein kinase C and CaM-kinase I1 phosphorylated the same site.

CaN' is a Ca2+/CaM-dependent protein phosphatase com-

* Supported in part by National Institutes of Health Grant NS 27037. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

3 Present address: the First Dept. Internal Medicine, Faculty of Medicine, Tokyo University, 7-1 Hongo 3-Chome, Bunkyo-ku, Tokyo 113, Japan.

To whom correspondence should be addressed Dept. of Molecu- lar Physiology and Biophysics, 702 Light Hall, Vanderbilt University Medical School, Nashville, TN 37232-0615.

The abbreviations used are: CaN, calcineurin; CaM, calmodulin; CaM-kinase 11, Ca2+/calmodulin-dependent protein kinase II; protein kinase C, Ca'+/phospholipid-dependent protein kinase; EGTA, [eth- ylenebis(oxyethylenenitrilo)]tetraacetic acid; HEPES, 4-(2-hydroxy- ethyl)-1-piperazineethanesulfonic acid; HPLC, high performance liquid chromatography; PS, phosphatidylserine; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

posed of two polypeptides, A and B, with molecular sizes of 58-61 and 19 kDa, respectively (1-3). The A subunit contains the CaM-binding (4, 5) and catalytic domains (6, 7), whereas the B subunit binds 4 eq of Ca2+ with high affinity (8, 9). Although physiological substrates for CaN have not been firmly established, the enzyme catalyzes dephosphorylation of not only phosphoserine/phosphothreonine but also phos- photyrosine (1-3,lO-12) under in vitro conditions, suggesting that CaN may have a rather wide physiological and regulatory role.

CaN has been purified from bovine brain (5, 13), bovine heart (14,151, rabbit skeletal muscle (16), and human platelets (17). Based on immunological data (18), specific protein phosphatase assays (19), and enzyme purification data, CaN has been thought to be significantly enriched in brain and skeletal muscle relative to other tissues. Purified CaN contains up to 0.6 eq of endogenous phosphate/mol of A subunit (ZO), suggesting that CaN may be regulated by phosphorylation. Re- cently, we showed (21) that CaN is phosphorylated by the autophosphorylated form of CaM-kinase 11. This phosphorylation is blocked when Ca2+/CaM is bound to CaN, suggesting interaction between the phosphorylation site and the CaM-binding domain. Although this phosphorylation does not appear to affect the binding of Ca2+/CaM to CaN, it does result in partial inactivation of CaN due to an increase in K , for protein substrates. In this paper, we have identified the regulatory phosphorylation site and localized it relative to the CaM-binding domain. Furthermore, we have also shown that this same site can also be phosphorylated in vitro by protein kinase C, which, like CaM-kinase 11, is very abundant in brain (22-24).

EXPERIMENTAL PROCEDURES

Materials-CaN (20), a gift from Dr. Marita M. King (Ohio State University), and CaM (25) were purified from bovine brain. Protein kinase C (26) and CaM-kinase I1 (27) were purified from rat brain by Dr. Roger J. Colbran (Vanderbilt University). The catalytic subunit of CAMP-dependent protein kinase (28) and the catalytic subunits of protein phosphatase I (29) and pig brain phosphatase IIA (30) were kindly provided by Drs. Jackie D. Corbin (Vanderbilt University), Balwant S. Khatra (California State University at Long Beach), and Shiaw-Der Yang (National Tsing Hua University, Republic of China), respectively. Rabbit skeletal muscle myosin light chain and myosin light chain kinase were generous gifts from Drs. Edwin G. Krebs and Peter J. Kennelly (University of Washington). [Y-~'P]ATP was prepared (31) using carrier-free 32P04 from ICN Pharmaceuticals. Syn- tide-2, a peptide substrate of CaM-kinase 11, was synthesized (24). Other materials were obtained as follows: PS, diolein, trypsin, and thermolysin, Sigma; cyanogen bromide and X-Omat RP film, East- man; phosphocellulose paper, Whatman; Sephadex G-25 (fine) and G-15, Pharmacia LKB Biotechnology, Inc.; and Millipore filters (0.45 pm), Nihon Millipore Kogyo K. K. (Japan).

Phosphorylation, Dephosphorylation, and Activity Assay of CaN- PS (0.2 mg) and diolein (0.02 mg) in chloroform were dried under a

16524

Regulation of Calcineurin by Phosphorylation 16525

stream of N,, suspended in 200 p1 of HzO, and sonicated with a microtip sonicator (Brow:n Sonifier, cell disruptor 185) using two 8-s bursts (output control = 2 ) at 4 "C. Phosphorylation of CaN (4-15 FM) by protein kinase C (0.43 PM) was performed in a 10-pl reaction containing 50 mM HEPES (pH 7.5), 0.1 mM [Y-~'P]ATP, 10 mM magnesium acetate, 0.4 mM CaCl,, 1 mg/ml bovine serum albumin, and dispersed lipids (0.1 :mg/ml PS and 0.01 mg/ml diolein). A t the desired times, aliquots were spotted on phosphocellulose papers (32) or added to sample buffer (2% sodium dodecyl sulfate, 750 mM 2- mercaptoethanol) for SD8-PAGE according to Laemmli (33). Phos- phorylation of CaN by the autophosphorylated form of CaM-kinase 11, activity assay of CaN, and dephosphorylation of [3ZP]CaN were performed as described previously (21).

Proteolytic Digestion and Analysis of Phosphopeptides-The phosphorylation reaction containing t3*P]CaN was subjected to proteolysis, diluted with 0.1% trifluoroacetic acid, and applied to a disposable C18 reverse-phase cartridge (Burdick & Jackson Laboratories Inc.). Free [-Y-~*P]ATP was removed by washing with 0.1% trifluoroacetic acid, and the 3ZP-peptides were eluted by a stepwise wash with 70% acetonitrile in 0.1% trifluoroacetic acid and concentrated using a Speed-Vac. In some experiments, the phosphorylation reaction mixture was first subjected to SDS-PAGE, and the 60-kDa 3ZP-subunit of CaN was extracted and subjected to proteolysis. Proteolysis was conducted at 30 "C for 16 h with 1 mg/ml trypsin in 0.1 M potassium phosphate (pH 8) or at rt9orn temperature for 16 h with 0.12 g/ml cyanogen bromide in 70% formic acid. The 32P-peptides were analyzed by reverse-phase HPLC 011 a Beckman CI8 Ultrasphere ODS column equilibrated in 0.1% trifluoroacetic acid and eluted with a gradient of acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 1 ml/min. Fractions of 1 ml were coll'ected, and their Cherenkov radiations were determined. Greater than ;35% of applied radioactivity was recovered from the column during the gradient. For localization of the 32P- residue, the 32P-peptides were subjected to automated Edman degra- dation in a Beckman Model 890C Sequencer; and at each cycle, the

release of 32P0, was determined. For determination of the peptide sequence, an Applied Biosystems Gas-Phase Sequencer was used.

Other Methods-Identification of 32P-amino-acids was determined by partial acid hydrolysis (6 N HCl for 2 h at 100 "C), high voltage paper electrophoresis at pH 1.9 and 2500 V for 1 h (34), autoradiog- raphy, and liquid scintillation counting. Concentrations of protein kinase C and CaM-kinase I1 were determined by the Bradford assay (35) using bovine serum albumin as standard. Concentrations of CaN and CaM were determined spectrophotometrically using absorbance indices for 1% protein solutions of 9.3 at 279 nm (36) and of 1.8 at 276 nm (37), respectively.

RESULTS

Identification of Phosphorylation Site-CaN, 32P-labeled to a molar stoichiometry of about 1 using the autophosphorylated form of CaM-kinase I1 (21), was cleaved with cyanogen bromide; and the digest was subjected to reverse-phase HPLC (Fig. lA). The single 3ZP-peptide, when subjected to automated Edman sequence analysis, exhibited a burst of 32P04 release at cycle 5 of the sequence (Fig. lA, inset). The cyanogen bromide 3ZP-peptide was subdigested with trypsin and chromatographed on Sephadex G-15 and then by reverse- phase HPLC. One sharp 32P-peptide and corresponding absorbance (210 nm) peak were detected (Fig. 1B). When the tryptic 32P-peptide was subjected to Edman analysis, 32P04 release occurred at cycle 3 (data not shown). Amino acid composition of the tryptic peptide indicated a rather hydro- phobic composition (Table I), and gas-phase sequence analysis yielded the sequence Val-Phe-Ser-Val-Leu-Arg.

Phosphorylation of CaN by Protein Kinase C-When CaN (5 p ~ ) was incubated with protein kinase (0.43 p ~ ) in the

FIG. 1. Purification ~P~~P- labe led peptide obtained by digestion of phosphorylated CaN. CaN (4 nmol) was phosphorylated by the autophosphorylated form of CaM-kinase I1 (33 pmol) in the presence of CaM ((45 pmol) as described (21). A t 5 min, the reaction was stopped by addition of excess EDTA, and t3'P]CaN was digested with CNBr and loaded onto a reverse-phase HPLC column (A) . Fractions at retention times of 43-47 min were collected. Part of the [32P]CaN was subjected to Edman sequence analysis for 32P04 release (A, inset). The remainder was redigested in 0.1 mM potassium phosphate (;pH 8) with 1 mg/ml trypsin at 30 "C for 30 min, and the 32P-peptide was further purified on Sephadex G-15 (bed volume of 50 ml; data not shown) and then by reverse- phase HPLC (B) .

0 5 10 15 20 25 30 35 40 45 50 55 I RETENTION TIME ( ain 1

-50

I/ 0.0 -

0 5 1 0 1 5 2 0 2 5 i 30 -D 35 40 45 50 55 60

RETD(TI0N TIME ( min 1

16526 Regulation of Calcineurin by Phosphorylation TABLE I

Amino acid composition of phosphopeptide of CaN obtained by digestion with cyanogen bromide and then trypsin

The mixture of fractions 37 and 38 in Fig. 1B was analyzed using a Waters Pico-Tag analyzer. Amino acids not shown were less than 10 pmol. The numbers in parentheses are the relative numbers of residues assumed to be present in the phosphopeptide.

Amino acid pmol

Ser G ~ Y Arg Ala TY r Val cys Leu Phe

101.41 (1)

117.58 (1) 21.27

14.34 26.47

11.0 234.25 (2)

138.90 (1) 117.22 (1)

1.00 , 1

43

0.25r I / LANE 1 2 3 4 5 6

INCUBATION(min) 5 10 20 30 30 30 Q

0 5 10 15 20 25 30 INCUBATION TIUE B i n )

FIG. 2. Phosphorylation of CaN by protein kinase C. CaN (5 p ~ ) (0, 0, lanes 1-5) or CaN storage buffer (lane 6) was incubated in 50 mM HEPES (pH 7.5),10 mM magnesium acetate, 0.4 mM CaC12, 1 mg/ml bovine serum albumin, 0.1 mM [y-"P]ATP, dispersed lipids (0.1 mg/ml PS and 0.01 mg/ml diolein), and protein kinase C (0.43 p ~ ) in the absence ( 0 lanes 1-4, and 6) or in the presence ( 0 lane 5) of 6 p~ CaM. At the indicated times, aliquots were spotted on phosphocellulose papers to determine phosphorylation stoichiometries or added to Laemmli sample buffer (33) for SDS-PAGE and autoradiogram. Molecular sizes are indicated (in kilodaltons) in the inset.

0 10 20 30 40 RTD(TI0N T I E bin)

FIG. 3. Analysis of tryptic phosphopeptides. [32P]CaN phosphorylated by protein kinase C (-) or CaM-kinase I1 (. . . . . ) was subjected to SDS-PAGE. The 60-kDa subunit protein extracted from the gel piece was digested with 1 mg/ml trypsin at 30 "C for 16 h and analyzed by reverse-phase HPLC. Samples of 1 ml were counted for Cherenkov radiation. The inset shows Edman sequence analysis of 32P0, release for peptides phosphorylated by protein kinase C (0) and the autophosphorylated form of CaM-kinase I1 (0).

TABLE I1 Effect of phosphorylation on phosphatase activity of CaN

CaN (4 p ~ ) was incubated in 50 mM HEPES (pH 7.5), 10 mM magnesium acetate, 0.4 mM CaCI2, 1 mg/ml bovine serum albumin, dispersed lipids (0.1 mg/ml PS and 0.01 mg/ml diolein), and protein kinase C (0.43 p ~ ) in the absence (nonphosphorylated) or in the presence (phosphorylated) of cold 0.1 mM ATP for 20 min. The phosphatase activities of nonphosphorylated and phosphorylated CaN (80-130 nM) were assayed in 50 mM HEPES (pH 7.5), 0.4 mM CaCI2, 1.1 p M CaM, 1 mM MnC12, 1 mg/ml bovine serum albumin, and varying concentrations (5-30 p ~ ) of D2P-labeled myosin light chain. Reactions were init.iated with addition of CaN. Aliquots were mixed with trichloroacetic acid (final concentration of 20%) at 1, 2, and 3 min and put on ice for a t least 10 min after addition of bovine serum albumin to 2.5 mg/ml. Following centrifugation, radioactivity in the supernatant was measured. Values are the mean 2 S.E. ( n = 5).

CaN phosphatase activity

Nonphosphorylated Phosphorylated

K, V,. K, V,. PM pmol/min/mg P M pmol/rnin/mg

9.1 2 0.1 0.260 f 0.035 17.5 f 0.1 0.264 f 0.024

presence of Ca", M e , [y-32P]ATP, PS, and diolein, the 60- kDa subunit of CaN was phosphorylated to stoichiometries of 0.9-1.0 within 20 min (Fig. 2). This phosphorylation was not observed if PS and diolein (data not shown) or protein kinase C was omitted from the reaction. Phosphoamino acid analysis revealed that greater than 95% of the radioactivity was on serine (data not shown). The inclusion of CaM (6 PM) in excess of CaN effectively inhibited the phosphorylation reaction (Fig. 2). The inhibition by CaM appears to be substrate-directed, i.e. due to the interaction of Ca2+/CaM with CaN, since phosphorylation of a synthetic peptide, syntide-2, by protein kinase C was not affected by 6 PM Ca2+/CaM (data not shown). There was no detectable phosphorylation of the 19-kDa subunit of CaN.

The 60-kDa 3 2 P - s ~ b ~ n i t was extracted by SDS-PAGE and subjected to digestion for 16 h with 1 mg/ml trypsin. Peptide mapping by HPLC showed only a single 32P-peptide which eluted at about 30% acetonitrile (Fig. 3). When the tryptic "P-peptides derived from CaN "P-labeled with either protein kinase C or CaM-kinase I1 were mixed and subjected to HPLC, they coeluted (data not shown). Analysis of these tryptic "P-peptides by sequential Edman sequencing gave

32P04 release at cycle 3 (Fig. 3, inset). Subdigestion of these two tryptic 32P-peptides with thermolysin resulted in identical shifts in the HPLC elution patterns (data not shown).

Effect of Phosphorylation on Phosphatase Activity-CaN was phosphorylated by protein kinase C to stoichiometries of 0.9-1.0 as described in Table 11. Nonphosphorylated CaN (control) was prepared by omission of ATP from the reaction. The protein phosphatase activity of CaN was then measured using varying concentrations of 32P-labeled myosin light chain. Phosphorylation of CaN increased the K, from 9.9 f 1.1 to 17.5 * 1.1 PM, but had no significant effect on the V,.. .

Dephosphorylation of f2P]CaN-Since CaN is a protein phosphatase, it was determined whether it catalyzed its own autodephosphorylation. In preparing ["PICaN, a special pre- caution was taken to ensure that most of the 32P04 was in CaN. The phosphorylation reaction mixture in the absence of CaN but in the presence of protein kinase C was incubated with cold ATP for 15 min prior to addition of CaN and [y- 32P]ATP. CaN phosphorylation was continued for an additional 30 min to a stoichiometry of about 1. [y-32P]ATP was removed by gel filtration to prevent possible CaN rephos-

Regulation of Calcineurin by Phosphorylation 16527

N W U R RATIOS OF Call TO CaN

FIG. 4. Effect of Ca"+ and CaM on CaN dephosphorylation. [32P]CaN was prepared as follows. First, protein kinase C (0.43 pM) was incubated in 50 mM HEPES (pH 7.5),10 mM magnesium acetate, 0.4 mM CaC4, 1 mg/ml bovine serum albumin, 0.1 mM cold ATP, and dispersed lipids (0.1 mg/ml PS and 0.01 mg/ml diolein) for 15 min. Then, 5 p~ [T-~'P]ATP and 15 p M CaN were added, and the reaction was conducted for an additional 30 min. [T-~'P]ATP was removed by gel filtration on Sephadex G-25. Dephosphorylation of CaN (0.35 p ~ , 1 mol of 32P04/mol of CaN) was determined in 50 mM HEPES (pH 7.5), 0.1 mhf EDTA, 1 mg/ml bovine serum albumin in the presence of varying cmoncentrations of CaM (0-6 pM) and 0.4 mM CaC12 (0) or 0.1 mM EGTA (0). Reactions were initiated with addition of [32P]CaN, and aliquots were spotted on phosphocellulose papers. The phosphatase activity is expressed as a percentage of the 32P04 release during the 1.0-min assay. Values are the mean k S.E. (n = 3).

phorylation by protein kinase C during the phosphatase assay reaction. In this preparation, only 2% of radioactivity was in the 80-kDa protein kinase C based on analysis by SDS-PAGE. The limited autodephsosphorylation observed in this preparation required Ca2+ and was stimulated by low concentrations of CaM (Fig. 4). However, concentrations of CaM at a molar ratio to CaN greater than 1.7 increasingly inhibited autodephosphorylation. This biphasic response of autodephosphorylation to varying concentrations of Ca2+/CaM was identical to that observed when the phosphorylation of CaN was catalyzed by CaM-kinase 11 (21) and suggests that autodephosphorylation was mainly intermolecular. At low concentrations, binding of Ca2+/CaM to one molecule of CaN would stimulate its activity toward dephosphorylation of another molecule of CaN which did not have Ca2+/CaM bound. Inhi- bition at high concentrations is probably due to the fact that binding of Ca2+/CaM t'o all the CaN sterically blocks dephosphorylation. This interpretation is consistent with the close proximity of the CaM-binding domain to the site of phosphorylation. Because the rate of autodephosphorylation was very low (0.18 nmol/min/mg, plus Ca2+ minus CaM), effects of other protein phosphatases were examined. Protein phosphatase I and phosphatase IIA catalyzed dephosphorylation of CaN at rates of 0.0107 and 0.308 pmol/min/unit of phosphorylase phosphatase activity, respectively. As observed with the autodephosphorylation experiments (Fig. 4), both dephosphorylation reactions were stimulated by Ca2+ and strongly inhibited by 6 p~ CaM (data not shown).

DISCUSSION

Recently, Kincaid et al. (38) published the sequence of the 307 C-terminal amino acids of CaN and identified the putative CaM-binding domain. We believed that the site of phosphorylation was within this Dortion of CaN since limited

proteolysis of [32P]CaN by trypsin resulted in a 43-kDa poly- peptide devoid of 3zPo4 (21) and of CaM binding [7, 39). Recent studies (41) on CaN establish that this limited proteolysis occurs at the C terminus and removes the CaM-binding domain. Knowledge of the consensus sequence for sites phosphorylated by CaM-kinase I1 (Arg-X-X-Ser/Thr; Ref. 42) allowed us to identify the sequence -Met-Ala-Arg-Val-Phe- - Ser-Val-Leu-Arg-Glu-Glu-Ser-Glu-Ser-Val-Leu-Thr-Leu- Lys-Gly-Leu-Thr-Pro-Thr-Gly-Met- (residues 192-217 in Ref. 38) as the only consensus sites for phosphorylation. This tentative assignment was attractive since Ser-197 and Ser- 203 would be positioned on the C-terminal boundary of the putative CaM-binding domain (assigned to residues 177-200). Thus, it would not be surprising that binding of Ca2+/CaM might block phosphorylation of either of these 2 serines, as was observed for phosphorylation of CaN by CaM-kinase I1 (21).

Determination of the site of phosphorylation was obtained by analysis of 32P04 release at cycle 5 of the cyanogen bromide 32P-peptide (cleavage C-terminal to Met) and cycle 3 of the tryptic 32P-peptide. Since there was no release of 32P04 at cycle 11 from the cyanogen bromide 32P-peptide, this indicated that only Ser-197, and not Ser-203, was phosphorylated. Although this analysis of 32P04 release strongly suggested Ser-197 as the unique site of phosphorylation, we could not be certain of this conclusion since the entire sequence of CaN was not published. Quantitative subdigestion of the cyanogen bromide 32P-peptide required high concentrations of trypsin (1 mg/ml), probably due to 2 Glu residues C-terminal to Arg- 200. Elution of the tryptic 32P-peptide at about 30% acetonitrile suggested a rather high degree of hydrophobicity for a peptide of only six amino acids, consistent with the predicted peptide from residues 195 to 200. Therefore, we decided to subject the tryptic 32P-peptide to gel filtration to remove contaminating larger tryptic fragments which would elute at this relatively high concentration of acetonitrile. This strategy was successful in that, during subsequent reverse-phase HPLC, the contaminating tryptic fragments coeluting from gel filtration eluted at a lower acetonitrile concentration, and a single absorbance peak was detected with the 3ZP-peptide which gave the correct amino acid composition. Subsequent amino acid analysis confirmed the sequence as Val-Phe-Ser- Val-Leu-Arg.

Although the phosphorylation of CaN is blocked by binding of Ca2+/CaM, [32P]CaN is still activated by Ca2+/CaM and binds to CaM-Sepharose (21). We were unable to detect an effect of this phosphorylation on the concentration required for half-maximal activation of CaN, but this question needs to be explored in a more quantitative manner. However, several other CaM-binding proteins are known to have phosphorylation sites, some of which affect binding of CaM. There are two CAMP-dependent protein kinase-catalyzed phosphorylation sites in smooth muscle myosin light chain kinase, one of which is within the CaM-binding domain; and phos- phorylations at both sites cause decrease in affinity for binding of Ca2+/CaM (43). Likewise, CaM-kinase I1 has a t least two regulatory autophosphorylation sites. Phosphorylation of Thr-286 (44), which is within the autoinhibitory domain just N-terminal to the CaM-binding domain (45-47), converts the kinase to the partially Ca2+-independent form (27, 48-51). Removal of CaM from this form of CaM-kinase I1 promotes additional Ca2+-independent autophosphorylation, probably at either Thr-305 or Thr-306 within the CaM-binding domain (52), which completely blocks binding of Ca2+/CaM (53). The 60- and 63-kDa isozymes of brain CaM-dependent cyclic

" nucleotide phosphodiesterases are specifically phosphorylated

16528 Regulation of Calcineu

by CAMP-kinase (54) and CaM-kinase I1 (55), respectively, resulting in decreased affinity for Ca2+/CaM. Phosphorylation of neuromodulin (also known as P-57, GAP-43, B-50, and F- 1) by protein kinase C inhibits the binding of CaM, which occurs in a Ca2+-independent manner, to this membrane protein (56).

The presence of Arg residues in close proximity on not only the N-terminal but also the C-terminal sides of the phosphorylated serine in CaN suggested to us that this site may also be phosphorylated by protein kinase C (57). Indeed, CaN was rapidly and stoichiometrically phosphorylated by protein kinase C. This phosphorylation reaction exhibited all the same features as phosphorylation by CaM-kinase 11: 1) inhibition by binding of Ca2+/CaM to CaN; 2) phosphorylation resulting in an increase in K,,, for myosin light chain with no change in VmaX; and 3) low rate of autodephosphorylation of [32P]CaN, but rapid dephosphorylation by protein phosphatase IIA >> phosphatase I. Proteolytic 32P-peptide mapping studies confirmed that protein kinase C phosphorylated the same site as CaM-kinase 11. Our results are at variance with those reported by Lim Tung (58), who reported phosphorylation of CaN (0.5-1.0 mol) by protein kinase C with no effect on phosphatase activity using [32P]casein as substrate. Details of the kinetic analysis were not given, but since [32P]casein is not a very good substrate for CaN (3), it may be difficult to reliably determine an increase in the K,. We also examined phosphorylation of CaN by CAMP-dependent protein kinase (0.25 PM) and found rather slow and substoichiometric phosphorylation (0.2-0.4 mol eq after 60 min) which was completely inhibited in the presence of excess CaM.' Proteolytic 32P-peptide mapping by HPLC indicated that the site of phosphorylation was the same as for CaM-kinase 11. Casein kinase I can also catalyze incorporation of up to 1.5 mol of phosphate into CaN, with no detectable effect on phosphatase activity (59).

Purified CaN contains up to 0.6 eq of phosphate (20), suggesting that phosphorylation of CaN occurs in vivo. How- ever, the site of this endogenous phosphate has not been determined. Phosphorylation and partial inactivation of CaN in vivo could enhance phosphorylation of those proteins nor- mally dephosphorylated by CaN such as microtubule-associ- ated protein 2 , ~ protein, myelin basic protein, tubulin, myosin light chain, protein phosphatase inhibitor 1, the a subunit of phosphorylase kinase, etc. (1-3, 60). Some of these proteins are also substrates for CaM-kinase I1 (61) and for protein kinase C (40). Experiments are in progress to examine incorporation of phosphate into CaN in culture brain cells and effects of agonists which act through diverse transmembrane signaling pathways.

Acknowledgments-We wish to thank Dr. Marita M. King for helpful discussions and Dr. Randall L. Kincaid for making the partial sequence of CaN available to us prior to its publication.

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Regulation of Calcineurin by Phosphorylation · Regulation of Calcineurin by Phosphorylation 16525 stream of N,, suspended in 200 p1 of HzO, and sonicated with a microtip sonicator