Phosphorylation of caseins and lactalbumin by ChlPKl and ChlPKz. Protein kinase activity of ChlPKl (4 pg) and ChlPK2 (6 pg) was measured with 100 pg of ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 257, No. 20, Issue of October 25, pp. 12157-12160. 1982 Printed In U.S.A.
Protein Kinasesfrom Spinach Chloroplasts 11. PROTEIN SUBSTRATE SPECIFICITY AND KINETIC PROPERTIES* (Received for publication, April 9, 1982)
Hector A. LuceroS, Zhe-F'uLing, and Efraim Racker From the Section of Biochemistry, Molecular & Cell Biology, Division of Biological Sciences, Cornell University, Ithaca, New York i4853
Two protein kinases (chloroplast protein kinases 1 and 2 (ChlPK, and ChlPK2))isolated from spinach chloroplast showed distinct differences in protein substrate specificity when a series of individual caseins, histones, and lactalbumins were tested. With casein as substrate, the V,, for ChlPK, was 800 picomoles X min" X mg of protein", and 300 picomoles x min" X mg of protein" for ChlPK2. Several tri- and dinucleotides (GDP, GTP, ADP, ITP) competed with ATP for ChlPK2, whereas only ADP inhibited significantly the protein kinase activity of ChlPKl with ATP as substrate. M&+ was required for bothkinases but some other divalent cations (Mn2+,Co2+)were almost as effective. Theoptimum pH was 6.5 for ChlPK, and 8.0 for ChlPK2.The activation energy was calculated to be 18,500 cal/mole for ChlPK1. C W K 2 showed a break in the Arrheniusplot with an activation energy of 32,400 cal/mole above 15 "C and 15,000 below 15 "C. The enzymes were rather resistant to N-ethylmaleimide. ChlPK, was inhibited about 30% at 10 m~ N-ethylmaleimide. N-Benzylmaleimide was more effective and inhibited both enzymes about 40% at 10 m~ after 30 min of incubation at 0 "C. ATP protected against inhibition by both N-ethylmaleimide and N-butylmaleimide.Both protein kinases were inhibited by low concentrations of CdC12. ChlPKl was autophosphorylated and also phosphorylated the small subunit of ribulose bisphosphate carboxylase. Neither ChlPK, nor CWK2 phosphorylated the lightharvesting complex. Crude fractions from spinach chloroplasts contained a protein kinase activity which phosphorylated (in the presence of [y3'P]ATP) a band which co-migrated with the light-harvesting complex in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ~
~~
~
At least 20 polypeptides between 5,000 and 70,000 daltons have been shown to be phosphorylated in chloroplast thylakoid membranes (1). In chloroplasts from peas (2) and from spinach (3), two major phosphorylated bands were seen after exposure to light. One was a 26,000-dalton polypeptide which co-migrated with the light-harvesting chlorophyll a/b-binding protein and the second was a 9,000-dalton polypeptide which co-migrated with the dicyclohexylcarbodide-binding protein of the ATPase complex. In the dark, the former was * This investigation was supported by Grant PCM80-21201, awarded by the National Science Foundation. 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. Recipient of a Fellowship from Consejo Nacional de Investigaciones Cientificas y Tecnicas from Argentina. 8 Supported by the Chinese government.
*
dephosphorylated much more rapidly than the latter (4). Threonine was reported to be the amino acid residue phosphorylated in the light in pea chloroplasts (2). In the preceding paper (5), we have shown that in spinach chloroplasts both serine and threonine residues were phosphorylated and we have isolated from these chloroplasts two distinct serine protein kinases after extraction with detergents. In SDS'-polyacrylamide gel electrophoresis, each purified protein appeared asa major band, ChlPKl corresponding to 25,000 daltons and ChlPK2 corresponding to 38,000 daltons. Both protein kinases phosphorylated serine residues of casein and histone 111s.In this paper, we describe the kinetic properties, sensitivity to inhibitors, and protein substrate specificity of these two enzymes. We also report on the phosphorylation of serine residues of endogenous substrates by a purified preparation of spinach protein kinase and by the catalytic subunit of CAMP-dependent protein kinase. On addition of the light-harvesting protein complex to an ammonium sulfate precipitate obtained from spinach chloroplast, a band comigrating with the light-harvesting protein became phosphorylated. EXPERIMENTALPROCEDURES
Materials-The various samples of milk proteins were generous g&s from Dr. E.W. Bingham and Dr. M. L. Groves (Eastern Regional Research Center, Philadelphia, PA). Phosvitin, casein (partidy hydrolyzed and dephosphorylated),histone I1 AS,histone IIIS, N-ethylmaleimide, and all the nucleotides were purchased from Sigma. NBenzylmaleimide was from ICN Pharmaceutical, Inc., Plainview, NY. Protamine sulfate was from Elanco Products Corp., Indianapolis, IN. Bio-Beads SM-2 were from Bio-Rad Laboratories, Richmond, CA. Methods-The preparation of ChlF'Kl and ChlPK2, protein kinase assay, autoradiography, and phosphoamino acid analysis were performed as described in the preceding paper (5).Purification of ribulose bisphosphate carboxylase (6), ferredoxin NADP reductase (7), CF, (8), andlight-harvesting protein (9)was as described in the references. Triton X-100was removed from the purified protein with Bio-Beads SM-2 (10). RESULTS
Substrate Specificity-A comparison of the kinase activities of ChlPKl and ChlPKz with over 20 samples of casein (from different cows) showeda distinct pattern of specificity. A few examples are given in Table I. The most striking difference was noted with a sample of /3 casein (Ad which was phosphorylated about 20 times better by ChlPK2 than by ChlPK1. In contrast, a sample of reduced and carboxymethylated a-lactalbumin was phosphorylated about 25 times more rapidly by ChlPK, than by ChlPK2. Histone 111, which is lysine-rich, was an excellent substrate for both enzymes while histone I1 was inactive. Phosvitin was a better substratefor ChlPKl thanfor ChlPK2. The abbreviations used are: SDS, sodium dodecyl sulfate; ChlPK1, chloroplast protein kinase 1; ChlPK,, chloroplast protein kinase 2; Mes, 4-morpholineethanesulfonic acid.
12157
Protein Kinases from Spinach Chloroplasts
12158
TABLE I Phosphorylation of caseins and lactalbumin by ChlPKl and ChlPKz Protein kinase activity of ChlPKl (4 pg) and ChlPK2 (6 pg) was measured with 100 pgof casein or lactalbumin as described in the accompanying paper under "Experimental Procedures" except that the protein kinase activity was measured at 37 "C for 30 min. ChlPK,
Substrate pmol 32PX min"
Experiment 1 aSl-caSein (B)430 BS1-casein(A') pSl-casein (A2) /3S1-casein (A3) K CaSeh 470 20 a-Lactalbumin (RCM)" 110 a-Lactalbumin Experiment 2 290 Casein (Sigma) Histone 11-AS 260111-S Histone Phosvitin Protamine sulfate
X
ChlPKz mgprotein"
780 860 lo00 1 990 510 110
TABLE I11 Effect of divalent cations on ChlPK, a n d ChlPK2 activity The assay mixture contained, in a final volume of 100 pl, 50 mM Mes/Tris (pH 6.5 for ChlPK1, pH 8.0 for ChlPKz), 500 pgof casein (Sigma), 4.4 pgof ChlPK, (or 2 pgof ChlPK2), 250 p~ [y3'P]ATP (specific activity, 208 cpm/pmol), and the indicated concentrations of EDTA or divalent cations. Reactions were carried out for 30 min at 22 "C,and analyzed as described under "Experimental Procedures." The values for the controls measured in the presence of 10 m~ MgC12 were: 190 and 50 picomoles of 32PX min" x mgof protein" for ChlPKl and ChlPK2, respectively.
330 370 20
Addition
Concentration mM
None EDTA MgCL
5
480 0
570 290 50
0
26
Relative activity ChlPK,
MnC12 34 29 coc12
80 50
RCM, reduced and carboxymethylated. 74
57
17
0.1 0.1 1.0 10.0 0.1 1.0 10.0 0.1 1.0 10.0
ChlPKz %
7 5 11
41 100
5 0 81 100
39 21 61
58 21 58
TABLE I1 Effect of nucleotides on protein kinase activities The protein kinase assay of ChlPKl (5 p g ) and ChlPK2 (5 pg) was carried out with casein as substrate as described under "Experimental Procedures" in the presence of 50 and 1 m~ concentrations of the nucleotides indicated in the table. The specific activities in the absence of added nucleotide were 180 picomoles of 32PX min" mg of protein" for ChlPKland 80 picomoles of 32PX min" X mgof protein" for ChlPK2. ChlPK, activity Additions
5 0 p nu-
1 m~ nu-
cleotide
cleotide
% control
100" Control 97 Control +73 AMP 4428 81 Control + ADP 111 93 Control + PPi 97 83 Control + GMP 37 86 Control + 95 GDP 100 Control + 87 GTP 100 Control + 91 CTP 115 98 Control +97 IMP 100 88 Control + ITP 107 Control +94 TTP 86 97 91 Control + UTP " Without second nucleotide.
ChlPK2 activity 5 0 nu-~ 1 m~ nucleotide cleotide 46 control
100" 93 82 95 85 75 88 91 98 85 92 104
112 85 87 50
77 57 87
Nucleotide Specificity-Both kinases used ATPasthe phosphorylating donor. None of the other tested nucleoside tri-, di-, or monophosphates (at a 50 PM concentration) inhibited, by either isotope dilution or competition, significantly the phosphorylation of casein by ChlPKl in the presence of [y-32P]ATP(Table 11).At 1 m ~ADP , inhibited both enzymes. ChlPK2,but not ChlPK1,was inhibited also by 50 PM GDP or GTP (25%and 12%,respectively) and by 1 m~ GDP or GTP (63%and 50%,respectively). ITP also inhibited at 1 m ~ . Cation Specificity-As shown in Table111, several divalent cations substituted for Mg2+with both ChlPKl and ChlPKz. Of particular interest is Mn2' which gavemaximal stimulation at 0.1 m~ with ChlPK1, but was not saturating at 10 m~ with ChlPKz. Ca2+and Fez+were tested, but no reliable data could be obtained because of visible precipitations occurring in the mixture. A t low concentrations, both seemed toactivate ChlPK, better than ChlPK2. Also of interest is the effectiveness of Co2+at 10 m~ concentrations as activator of both ChlPKl and ChlPKz.
FIG.1. pH dependence of ChlPK, a n d ChlPKz activities. Kinase activity of ChlPK, and ChlPK, was measured with Mes/Tris buffer as described under "Experimental Procedures" of Ref. 5, except that the pH was adjusted to thevalues indicated in the figure. Kinetic Properties of ChlPKl and ChlPKz-Both enzymes had an apparentK,,, for ATP of 25 PM. Vmaxwas 800 picomoles of 32PX min" X mg of protein" for ChlPKl and300 picomoles X min" X mg of protein" for ChlPK2. The pH optimum for ChlPK, was pH 6.5 and for ChlPKz pH 7.5-8.0 (Fig. 1). V,. was studied as a function of the temperature. The activation energy obtained from the datashown in Fig. 2 was calculated to be 18,500 cal/mole for ChlPK, and 32,400 cal/mole for ChlPKz above 15 "C and 15,000 below 15 "C. A t 10 m ~N-ethylmaleimide , inhibited ChlPKl about 30% but had no effect on ChlPKz. The inhibition of ChlPKl by N ethylmaleimide was prevented by 1 m~ ATP (Table IV). N Butylmaleimide dissolved in dimethyl sulfoxide inhibited both enzymes at 10 m~ concentration after incubation for 10-30 min at 0 "C (data notshown). A small decrease in activity by dimethyl sulfoxide alone was subtracted in the calculation of inhibition. The inhibition of ChlPKl by N-ethylmaleimide or of N-butylmaleimide was greater (approximately 75%) when tested at pH 8.0 rather than 6.5. Cd2+was a rather effective inhibitor of ChlPKz at 10 PM. Much higher concentrations of
Protein Kinases from Spinach Chloroplasts
12159
I
.2. .-C
I
n
I
PK7
\Ea
:18.500 cal per mol
FIG. 3. Phosphorylation of ribulose bisphosphate carboxylase b y ChlPKl and b y catalytic subunit of CAMP-dependent
k : 3 2 . 4 0 0cal pe In0
b 101
3.2
3.3
I
1
I
3.4
3.5
3.6
3.7
FIG.2. Activation energy of ChlPKl and ChlPKz. Kinase activity of ChlPK, and ChlPKz was measured as described under “Experimental Procedures” of Ref. 5, except that the samples were incubated a t the temperaturesindicated in the figure. TABLE IV Effect of N-ethylmuleimide on ChlPK activity; protection by ATP ChlPKl (4 pg) and ChlPKz (4 p g ) were incubated in a medium containing 50 m~ Mes/Tris (pH 6.5) for ChlPKl and pH 8.0 for ChlPKz, 10 m~ NaF, and, where indicated, 10 mM N-ethylmaleimide, 1 m~ ATP (containing 130 cpm/picomole of ”Pi) on ice for 10 min. Then, 30 pl of a mixture containing 500 pg of casein (Sigma), 20 mM MgClz, 250 p~ ATP (130 cpm/picomole of :r2PJ was added and incubated a t room temperature for 30 min. Protein kinase activity was measured as described under “Experimental Procedures” in Ref. 5. Treatment
bg$,,l$pmol-7 min-
Inhibition X
x mg protein - I
ChlPKl Control Control + N-ethylmaleimide Control + N-ethylmaleimide + ATP Control + ATP ChlPKz Control Control + 10 m~ N-ethylmaleimide
protein kinase. Samples containing 3 pg of ChlPKl (lune I), 3 pg of ChlPK, (lune 2), 15 pgof catalytic subunit of CAMP-dependent protein kinase (lune 3), 25 pg of ribulose bisphosphate carboxylase (lune 4), 3 pg of ChlPKl and 25 pg of ribulose bisphosphate carboxylase (lune 5), 3 pgof ChlPKz and 25 pg of ribulose bisphosphate (lune 6 ) , and 15 pg of catalytic subunit of CAMP-dependent protein kinase and 25 pg of ribulose bisphosphate carboxylase (lane 7) were incubated for 30 min a t room temperature in a medium (75 pl) composed of 50 m~ Mes/Tris (pH 6.5)for ChlPK,, pH 7.5 for catalytic subunit of CAMP-dependent protein kinase, and pH 8.0 for ChlPKz, 10 RIM MgC12, and 5 p~ [y3’P]ATP (2000 cpm/picomole). The reaction was terminated by addition of an equal volume of a buffer containing 50 m~ Tris-HC1 (pH 6.8),1% SDS, 2 mM EDTA, 0.5 M pmercaptoethanol, 10% glycerol, and 0.001% bromphenol blue. Samples were incubated a t 45 “C for 3 h, electrophoresed in SDS-polyacrylamide gel electrophoresis, and autoradiographed as described under “Experimental Procedures.” Roman numerals indicate the mobilities of the large subunit of ribulose bisphosphate carboxylase (I),catalytic subunit of CAMP-dependent protein kinase (ZZ), ChlPK, (ZZZ), ChlPK2 (ZV), small subunit of ribulose bisphosphate carboxylase (V), and the front of the gel (VI).A, Coomassie blue stain, B, autoradiograph.
s
250 180 250 250
0 29
90 90
0 0
0 0
FIG. 4. Phosphorylation of the lighbharvesting chlorophyllprotein complex by a crude ammonium sulfate fraction. Samples containing 10 pg of light-harvesting chlorophyll-protein complex CdC12 were required to affect a partial (33%) inhibition of (lune I), 5 pg of ammonium sulfate fraction from spinach chloroplasts (lune 2),and 10 pgof light-harvesting chlorophyll-protein complex ChlPKl (data not shown). and 5 pg of ammonium sulfate fraction(lune 3) were incubated for30 Both kinases were sensitive to detergents. ChlPKland min a t room temperature in a medium containing 50 m~ Mes/Tris ChlPK2 were inhibited about 55% at 30 m~ octylglucoside (pH 7.8), 10 m~ MgC12,lO m~ NaF, and 5 p~ [y3*P]ATP (2000cpm/ and 30% at 10 m~ octylglucoside. Sodium cholate at 0.1% picomole). The reaction was terminated and electrophoresed as deinhibited both enzymes about 25%and a significant inhibition scribed in the legend to Fig. 3. The arrow indicates the position of the light-harvesting protein complex. A, Coomassie blue stain; B, was seen even at 0.01% (data not shown). Phosphorylation of Endogenous Proteins Involved in Pho- autoradiograph.
tosynthesis-As can be seen from Fig. 3, ChlPKl phosphorylated itself as well as several minor impurities (B, lune I ) which were not detected by Coomassie blue staining (A,lane I ) . A band co-migrating with the small subunit of ribulose bisphosphate carboxylase became phosphorylated on exposure to ChlPKl (B,lune 5), whereas ChlPK, had no effect (B,
lane 6).The catalytic subunit of CAMP-dependent protein kinase phosphorylated itself and some minor impurity (B, lane 3), as well as a band corresponding to the mobility of the large subunit of ribulose bisphosphate carboxylase (B, lane 7). Minor phosphorylation bands were Seen with ChlPKl in
12160
Protein Kinases from Spinach Chloroplasts
ples of casein in exploring substrate specificity. As shown in Table I, a commercial preparation of casein, widely used for protein kinase assay, was phosphorylated almost equally well by ChlPKl and ChlPK2. Some casein samples, reduced and carboxymethylated a-lactalbumin andphosvitin, showed 3- to 20-fold differences in relative reactivity. ChlPKl responded specifically to adenine nucleotides, “P-tyrosine whereas ChlPK2 interactedwith other tri- anddinucleotides, particularly GDP. Rather surprisingly, CoC12 substituted for MgC12 and was much superior to MnC12. Several other divalent cations were tested (e.g. Fe2+and Ca2+), but the results -P-threonine were not recorded because of interference by the formation of precipitates. CdCh was found to be a potent inhibitor, partic-P-serine ularly of ChlPK2 (approximately 80% at 10 p ~ ) . We have no information about thephysiological role of the two serine phosphorylating enzymes in spinach chloroplasts. The only clue described in this paper is the phosphorylation FIG. 5. Phosphoamino acid analysis or’ casein phosphoryl- of the small subunit of ribulose bisphosphatase by ChlPKl. ated by ammonium sulfate fraction. Casein (250pg) was incubated Since the large subunit of this enzyme is catalytically active, for 30 min at 37 “C with the ammonium sulfate fraction (15 pg) in a the function of the small subunit was called a mystery in a medium containing 50 rn Mes/Tris (pH 7.8). 10 m M NaF, 10 mhf MgC12,5 PM [Y-~~PIATP (2000 cpm/picomole), and 50 m dithiothre- recent review (11).It may therefore be of considerable interest acids were to explore a possible regulatory effect of phosphorylation of itol. The reaction was terminatedandphosphoamino analyzed as described under “Experimental Procedures.” the small subunit on the catalytic properties of this important enzyme of photosynthesis. There is considerably more information and more speculathe region of the large subunit of ribulose bisphosphate carboxylase and with catalytic subunitof CAMP-dependent pro- tion about the possible role of phosphorylation of the lighttein kinase in the region of the small subunit of ribulose harvesting protein in photosynthesis (12).Neither ChlPKl nor ChlPK2 phosphorylated this protein. On the other hand, a bisphosphate carboxylase. partially purified preparation from spinach chloroplasts, The band corresponding to thelight-harvesting chlorophyllprotein complex was not phosphorylated by either ChlPKl or which contained threonine as well as serine phosphorylating ChlPK2 (data notshown). However, as shown in Fig. 4B (lane kinases, phosphorylated a band that co-migrated with the 3), it was phosphorylated by an ammonium sulfate fraction light-harvesting protein. Whether or notthis phosphorylation i l require exploration in refrom extracts of spinach chloroplasts (see Table I of Ref. 5). has physiological sigmficance w constituted systems of photophosphorylation. Moreover, it can be seen that the addition of highly purified light-harvesting complex (Fig. 4A, lane I ) to the crude fracAcknowledgments-We wish to thank Dr. R. E. McCarty and Dr. tion gives rise to multiple phosphorylation of contaminants M. Newman for a critical review ofthe manuscripts. not seen by Coomassie blue staining (compare Fig. 4A, lane REFERENCES 3, and Fig. 4 B , lane 3). Neither ChlPKl nor ChlPK2 phosphorylated ferredoxin NADP reductase. 1. Suss, K. H. (1981)Biochem. Biophys. Res. Commun. 102,724-729 With casein as substrate, the ammonium sulfate fraction 2. Bennett, J. (1977) Nature 269,344-346 3. Alfonzo, R., Nelson, N., and Racker, E. (1980) Plant Physiol. 65, phosphorylated serine as well as some threonine residues (Fig. 730-734 5). 4. Bennett, J. (1980) Eur. J.Biochem. 104,85-89 5. Lin, Z. F., Lucero, H. A., andRacker, E. (1982) J. Biol. Chem. DISCUSSION There are, as shown in this paper, at least three protein kinases in spinach chloroplasts that can besolubilizedby detergent extraction. Two of these were purified and shown to have distinctly different kinetic properties. The activity of ChlPKl exhibited surprisingly little response to pH in the range from 5-10 with a minor optimum peak at pH 6.5. In contrast, ChlPK2 showed a rather steep peak at pH 7.5-8.0. We have shown the usefulness of assaying individual sam-
257,12153-12156 6. Johal, S., and Bourque, D. P. (1979) Science 204,75-77 7. Zanetti, G., and Curti, B. (1980) Methods Enzymol. 69,250-255 8. Lien, S., and Racker, E. (1971) Methods Enzymol. 23,547-555 9. Burke, J. J., Ditto, C. L., andAmtzen, C. I. (1978)Arch. Biochem. Biophys. 187,252-263 10. Holloway, P. W. (1973)Anal. Biochem. 53,304-308 11. Lorimer, G.H. (1981)Annu. Reu. Plant Physiol. 32,349-383 12. Allen, J. F.,Bennett, J., Steinback, K. E., and Amtzen, C. J. (1981) Nature 291,25-29
