Planta

Planta (1989) 178:199-206

9 Springer-Verlag 1989

Characterization of a protein-kinase activity associated with phytochrome from etiolated oat (Arena sativa L.) seedlings* Rudolf Grimm 1' z, Doris Gast 1, and Wolfhart Riidiger 1 1 Botanisches Institut der Universit/it Mfinchen, Menzingerstrasse 67, D-8000 Mfinchen 19, and 2 Institut ffir Biologie II/Botanik der Universitfit Freiburg, Schfinzlestrasse 1, D-7800 Freiburg, Federal Republic of Germany

Abstract. A protein-kinase activity which is co-purified with phytochrome from etiolated oat seedlings was investigated in some detail. Whereas phytochrome was always phosphorylated in solution (together with some contaminating protein bands), radioactive phosphate was not found in the phytochrome band after native gel electrophoresis and incubation of the entire gel with labeled ATP. Since protein kinases are usually autophosphorylated under these conditions, the result shows that the kinase activity does not reside in the phytochrome molecule itself. Radioactivity was exclusively detected in a band with the apparent molecular weight 450 kDa; sodium-dodecyl-sulfate gel electrophoresis revealed an apparent molecular weight of 60 kDa for the phosphorylated subunit. The Nterminal amino-acid sequence A L E S A ~ K~ V P W was determined for this subunit which is a potential candidate for the protein kinase. The optimum conditions (pH, metal ion concentration) and kinetics of the phosphorylation reaction were determined. The presumed connection between proteinkinase activity and the signal chain leading from the far-red-absorbing form of phytochrome to physiological responses still awaits elucidation. Key words: Arena C a l m o d u l i n - Etiolated seedling - Phytochrome - Protein kinase - (ATP-dependent)

* Dedicated to Professor A. Trebst on the occasion of his 60th birthday Abbreviations: Bistris=2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-l,3-propanediol; kDa=kilodalton; Pfr=far-red absorbing form of phytochrome; Pr=red-absorbing form of phytochrome; PMBS =p-chloromercuribenzenesulfonate; SDS = sodium dodecyl sulfate; Tris = 2-amino-2-(hydroxymethyl)k1,3-propanediol

Introduction

Phytochrome is the main photoreceptor for the light-mediated development and differentiation of plants (Furuya 1987; Eilfeld and Haupt 1989). Whereas the biliprotein phytochrome itself has been characterized in considerable detail in its molecular properties and photochemistry, the signal chain which leads from phytochrome to differential expression of nuclear genes still awaits its elucidation. Differences in the protein conformation between the physiologically inactive form (Pr) and the active form (Pfr) have been demonstrated by several methods (Cordonnier etal. 1985; Jones et al. 1985; Lagarias and Mercurio 1985; Vierstra etal. 1987; Grimm et al. 1988). The differential phosphorylation of Pr and Pfr with mammalian protein kinases pointed to differential exposure of potential phosphorylation sites of the phytochrome molecule (Wong et al. 1986). These authors discussed also the possibility that phytochrome itself could be a protein kinase. This possibility was later corroborated by detection of a certain aminoacid sequence homology between phytochrome and known protein kinases (Lagarias et al. 1987). We were also able to detect protein-kinase activity in our phytochrome preparations. A more detailed investigation revealed, however, that the kinase activity does not reside in the phytochrome molecule itself. These experiments and a preliminary characterization of the kinase activity are described in the present paper. Material and methods Phytochrome and kinase preparation. Since the protein kinase co-purifies with phytochrome, the purification procedure follows exactly the protocol for isolation of phytochrome (124 kDa) from etiolated, 3.5-day-old oat seedlings (Arena sativa L., cv. Pirol; Baywa, Munich, FRG) (see Grimm and

200 Rfidiger 1986). After washing the phytochrome precipitate with 100 mM phosphate buffer, the purity index of phytochrome varied between A665/A280 =0.59 and 0.95 in the single preparations. The standard kinase assay contained the following components, unless otherwise stated: 2 gg phytochrome, 20 mM MgCl2, 10 gM ATP (3.7.1011Bq.mmol-1), 20raM 2-[bis(2hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol (Bistris) buffer, pH 7.2. Final volume was 20 lal. All experiments were performed under green safelight. The mixture was incubated at 30~ for 30 min, unless otherwise stated. The reaction was then stopped by addition of 20 gl sample buffer which contained 65 mM 2-amino-2-(hydroxymethyl)-l,3-propanediol(Tris)-HC1, pH 7.8, 5% (v/v) 2-mercaptoethanol, 2% (w/v) sodium dodecylsulfate (SDS), J5% (v/v) glycerol and 0.5% (w/v) bromophenol-blue. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out according to Laemmli (1970) on 10% gels. The gels were then stained with Coomassie Blue or, in some experiments, with silver nitrate. After destaining, the gels were applied to a film (X-OMAT AR; Kodak) and amplifier foil (MR 800, Agfa Geavaert, Leverkusen, FRG) for 15 h at - 7 0 ~ C. Radioactive bands were cut out of the gel and the radioactivity determined in a scintillation counter. Phosphorylation in the polyacrylamide gel. For "native" electrophoresis, phytochrome preparations or supernatants from the washing with 100 mM phosphate buffer (see under phytochrome and kinase preparation) were irradiated for 5 min with far-red ( = P r form) or red light ( = P f r form) and applied to a 7.5% polyacrylamide gel. Electrophoresis was carried out under green safelight for 16 h with a constant current of 12 mA in a system as described by Laemmli (1970) but without SDS and 2-mercaptoethanol. The gel was then washed 10 times for 10 rain with 10 mM Tris buffer, pH 7.2, containing 20 mM MgC12 and 10% glycerol. The washed gel Was then heat-sealed in plastic foil and incubated with 1.5 ml of the same buffer containing 5.66.106 Bq [732p]ATP for 30 min at 25 ~ C. The gel was then washed 10 times with methanol/acetic acid/water (4:1:5, by vol.) in order to fix the proteins in the gel and to remove the excess radioactivity. Coomassie-staining, destaining, drying and autoradiography were performed as described above. The desired protein bands were cut out of the gel and rehydrated for 20 rain with 125 mM Tris buffer, pH 6.8, containing 0.1% SDS. The gel pieces were then equilibrated with 50 mM Tris buffer, pH 6.8, containing 0.1% SDS, 14 mM 2mercaptoethanol, 5 mM ethylene diaminetetraacetic acid (EDTA) and 10% glycerol and applied to a 10% polyacrylamide gel. The SDS electrophoresis was carried out according to Laemmli (1970). Coomassie-staining, destaining, drying and autoradiography were performed as described above. Westernblot analysis for phytochrome was performed with the monoclonal antibody Z-3BI as described by Grimm et al. (1986). For determination of the N-terminal amino-acid sequence, the autophosphorylated protein band was cut out and treated as described above. After re-electrophoresis in the SDS-containing system, the protein bands were blotted onto a modified glass-fiber sheet (Eckerskorn et al. 1988), stained with Coomassie Blue and destained. The determination of phenylthiohydantoin amino acids in a glass-phase sequencer was performed as described earlier (Grimm et al. 1988).

Results

The first experiments on the in-vitro phosphorylation of phytochrome were performed in analogy

R. Grimm et al.: Phytochrome and protein kinase in oat seedlings

Fig. 1. Phosphorylation of 124-kDa phytochrome with and without histone and a protein kinase from the plastid envelopes of etiolated Arena seedlings. Phytochrome (final concentration 0.8 gM) was incubated in 20 mM Bistris buffer, pH 7.2, containing 20 mM MgC12 and 10.5 laM [7-32p]ATP (5.3.1012 Bq. mmo1-1) for 30 rain at 30 ~ C. The samples were applied to a 10% polyacrylamide gel. The autoradiogram after electrophoresis is shown here. Lane a: Pr form + kinase; b: Pfr form + kinase; c: Pr form (without kinase); d." Pfr form (without kinase); e: Pr form + histone + kinase; f : histone + kinase (without phytochrome); g: Pfr form + histone + kinase

to the experiments of Wong et al. (1986) but using a plant protein kinase isolated from pea chloroplast envelopes (Soll 1985, 1988) instead of mammalian protein kinases. The Pr and Pfr form of phytochrome were phosphorylated to about the same extent by the envelope kinase (Fig. 1, lanes a, b). This kinase apparently resembles the mammalian protein-kinase A but is clearly different from kinases C and G which react preferentially with the Pr form of phytochrome (Wong et al. 1986). To our surprise, omission of the envelope kinase preparation which was dissolved in 50% glycerol led to a higher incorporation of phosphate into the Pr form (Fig. 1, lane c); the Pfr from (lane d) was not phosphorylated under these conditions. Since our Pr sample also seemed to cause some additional phosphorylation of histone (Fig. 1, lane e, compared with lanes f, g) we decided to investigate the kinase activity of this preparation in more detail. Lagarias et al. (1987) had accumulated some evidence for phytochrome itself being a protein kinase. In order to check this possibility, we compared the phosphorylation of phytochrome at several purification steps (data not shown) and of the final phytochrome preparations. The eluate from the hydroxyapatite column always showed a very

R. Grimm et al. : Phytochrome and protein kinase in oat seedlings

Fig. 2. Phosphorylation of various preparations of 124-kDa phytochrome from etiolated Arena seedlings. For conditions, see Fig. 1. lane a: purity index A665/A2so =0.95; b: purity index A665/A28o=0.87; c.' purity index A665/A28o=0.72; d: purity index A665/A28 o = 0.59 Table 1. Phosphorylation of 124-kDa phytochrome preparations of varying purity from etiolated Arena seedlings. Incubation conditions are given in the legend of Fig. 1. Phytochrome bands were cut out of the gel and the radioactivity determined by scintillation counting. Values are the means of two independent experiments Purity index (A665/A28o)

Radioactivity of Pr

0.95 0.87 0.72 0.59

Pfr

cpm

%

cpm

%

96 196 288 584

100 204 300 608

67 96 160 371

100 143 239 554

high protein-kinase activity and many phosphorylated bands on SDS gels after incubation with labeled ATP. Most of this kinase activity could be removed by washing the phytochrome precipitate with phosphate buffer, the last step of our purification procedure (see Grimm and Rfidiger 1986). The remaining protein-kinase activity seemed to correlate inversely with the purity index of the particular sample (Fig. 2, Table 1). We found this relationship in freshly prepared samples as well as in samples which had been stored at - 2 0 ~ for various times. It is clearly evident from these experiments that the purest phytochrome preparations had nearly no kinase activity (Fig. 2 lane a) but that kinase activity seems to increase with the decreas-

201

ing purity index of the phytochrome preparation. The phosphorylation of the phytochrome band is summarized in Table 1. Under these conditions, several phosphorylated bands could be detected on the autoradiogram although no proteins were detectable with Coomassie-staining. Some of the contaminating protein bands could be detected with silver stain. One obvious explanation for this finding could be that the protein kinase is a contaminant of the phytochrome preparation. However, the alternative explanation, that an activator had been removed by the last purification step and that the kinase proper is phytochrome itself, could not be excluded from these experiments. Wong et al. (1986) described a kinase activity in their phytochrome preparations only in the presence of histone, polylysine or similar basic polymers. Although we could not confirm an increase of kinase activity caused by histone or polylysine in our phytochrome preparations, the eventual requirement for a natural activator could not yet be ruled out. We checked this possibility in the following experiment (Fig. 3). Pure phytochrome (A665/A2so = 0.99) and the last washing of the phytochrome precipitate with 100 mM phosphate were loaded onto a polyacrylamide gel for "native" electrophoresis. Phytochrome migrated with an apparent size of about 330 kDa, and the main contaminant in the washing migrated at about 450 kDa and at higher aggregate sizes (Fig. 3 A, lanes a-d). Incubation of the gel with [7-32p] ATP resulted in incorporation of label only into the 450-kDa band but not into the 330-kDa phytochrome band (Fig. 3 A, lanes eh). The same preparations yielded highly labeled phytochrome when the incubation with [732p]ATP was carried out in solution under otherwise identical conditions (data not shown, but see Figs. I, 2). In order to check the identity of the phosphorylated proteins, the labeled bands were cut out and applied to an SDS gel. For comparison, the phytochrome bands were also treated in the same way. Re-electrophoresis with SDS and staining with Coomassie Blue yielded the known 124-kDa bands of undegraded Pr and Pfr from the 330-kDa band and a single 60-kDa band from the 450-kDa complex (Fig. 3 B, lanes a-d). The autoradiogram of this gel showed radioactivity only in the 60-kDa band but not in the phytochrome band (Fig. 3 B, lanes e-h). Protein kinases can generally be detected via autophosphorylation. Nevertheless, we considered the possibility that phytochrome might catalyze phosphorylation of the 60-kDa protein without autophosphorylation. The 450-kDa complex should contain phytochrome in this case. Western-blot

202

Fig. 3A, B. Phosphorylation on a polyacrylamide gel of Arena phytochrome preparations containing a protein kinase. A Electrophoresis on a 7.5% gel under non-denaturating conditions. Arrows indicate the position of marker proteins: urease= 272kDa (monomer) and 544kDa (dimer), apoferritin= 443 kDa. After electrophoresis the gel was incubated with 30 nM [7-32p]ATP (11.1.107 Bq.nmo1-1) for 30 min at 25~ After washing ten times with methanol/acetic acid/water (4:1 : 5, by vol.), the gel was dried and applied to an X-ray film. The autoradiogram is shown in lanes e-h. Lane a = Pr (8 gg), b = Pfr (8 gg), e = supernatant from the last washing step of phytochrome with 100 mM phosphate, Pr form, d = identical with c, but Pfr form. Lanes e-h = autoradiogram of lanes a-d, respectively. B The phytochrome or radioactive bands, respectively, obtained under A, were cut out, rehydrated with 125 mM Tris buffer (pH 6.8) containing 0.1% SDS, 14 mM 2-mercaptoethanol, 5 mM EDTA and 10% glycerol and applied to a 10% polyacrylamide gel. Electrophoresis was carried out using the system of Laemmli (1970) containing 0.1% SDS. L a n e s ~ d show the Coomassie-staining, lanes e-h the autoradiogram. Lanes a, e = P r band of Fig. 3A, lane a," lanes b, f = P f r band of Fig. 3A, lane b; lanes c, g = radioactive band of Fig. 3A, lane c; lanes d, h = radioactive band of Fig. 3A, lane d

analysis did not reveal any phytochrome in the 450-kDa complex, however, either after blotting directly from the native gel or after re-electrophoresis with SDS. We confirmed, in this connection, that the monoclonal antibody Z-3B1, which binds to a peptide region near to the phytochrome chromophore (Grimm et al. /986), detects phosphorylated Pr and Pfr as well as non-phosphorylated phytochrome. It is known that reaction of phytochrome with ubiquitin results in high-molecular-

R. Grimm et al.: Phytochrome and protein kinase in oat seedlings

mass species (Shanklin et al. 1987). We could not detect any reaction, however, in our immunoblots with polyclonal anti-ubiquitin antibodies which had previously been used successfully for investigation of ubiquitin-phytochrome conjugates in Avena (Speth et al./987). The possibility that phytochrome gains kinase activity only after phosphorylation was tested in another set of experiments. The phytochrome preparations were phosphorylated at first with non-labeled ATP in solution as described above. For this reaction, both Pr and Pfr forms were used. The phosphorylated forms were then either used as such or phototransformed into the alternative forms before further use. All four samples were then subjected to "native" electrophoresis, incubated with [732p]ATP and analyzed in the same way as non-phosphorylated phytochrome (see Fig. 3). Identical results (data not shown) were obtained in each case: only the 60-kDa band was labeled in each sample whereas no label was found in the phytochrome band. In order to characterize the labeled 60-kDa protein further we determined its N-terminal amino-acid sequence. For this purpose, protein bands from the SDS gel were electroblotted onto activated glass-fiber sheets and directly applied to the gasphase sequencer (see also Grimm et al. /988). The following sequence was determined: A L E SA ~ K ~ V P W. Since the amino-acid exchanges at positions 5 and 7 were found in a ratio of about 1:1, the 60-kDa band must contain at least two isoproteins in about equal quantity. The occurrence of even more isoproteins is indicated by a further exchange of P-9 for K, and traces of F and N. These sequences belong, however, to only minor components of the entire complex. Another approach to identify the protein kinase in the phytochrome preparation was to drastically decrease the ATP concentration. For this series of experiments, a phytochrome preparation ( A 6 6 5 / A z s o = 0 . 8 9 ) w a s used in which no protein band was detectable after staining with Coomassie Blue except the 124-kDa phytochrome band (data not shown). Nevertheless, incubation with 15 nM ATP and I m M MgC12 resulted mainly in phosphorylation of a band at the position of the 60-kDa protein but not in the phosphorylation of phytochrome (Fig. 4, lanes a, b). Phytochrome was only phosphorylated at an MgC12 concentration of 3 m M or higher (Fig. 4, lanes c-f). Under these conditions, bands at 7, 18.5, 23, 29, 38, 45, 55 and 66 kDa were also phosphorylated; these were only labeled in traces at 1 m M MgC12. A similar effect to that with MgC12 was found

R. Grimm et al. : Phytochrome and protein kinase in oat seedlings

203

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0.2-

~,

/ /

%

"~

t

k

i

10

20

/,,0

mH

Fig. 5. Dependence of the phosphorylation of A r e n a phytochrome on metal ions. Conditions for the reaction were as in Fig. 1 except for the metal-ion concentration which was varied from 0 to 20 mM MnC12 (9169 or from 0 to 50 m M MgC12 ( [ ] - - n ) . The reaction mixture was subjected to SDS gel electrophoresis. The phytochrome band was cut out and the radioactivity determined by scintillation counting

Fig. 4. Phosphorylation of 124-kDa A r e n a phytochrome with carrier-free ATP. The phytochrome preparation (final concentration 0.8 ]aM) was incubated in 20 m M Bistris buffer, pH 7.2, containing 15nM [7-32P]ATP (11.1.1013 Bq.mmo1-1) and varying Mg concentrations for 30 min at 30~ C. The samples were then subjected to SDS gel electrophoresis. Lanes a, c, e = Pr; lanes b, d, f = Pfr; lanes a, b = 1 m M MgC12 ; lanes c, d = 3 mM MgCI2 ; lanes e, f = 5 mM MgCI2 ; phytochrome is only phosphorylated in lanes c - f but not in lanes a, b

~= E 0.t, ~= ~_ E ='0.2a.

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I-~176

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with CaC12 (data not shown). Divalent cations are known to increase among other effects - potential protein aggregation. These results indicate therefore that the 60-kDa protein which reacts even at low MgC12 or CaCI2 concentrations might be the autophosphorylated protein kinase. This is consistent with the results of Fig. 3. Phytochrome then belongs to the many "substrate" proteins which are phosphorylated only in the presence of divalent cations, i.e. under conditions in which there is a higher probability of a close contact between the kinase and the substrate protein. The requirement of phosphorylation for MgCI2 or MnC12 was investigated next. As shown in Fig. 5, the optimum concentration of MgC12 is 20 mM, that of MnC12 5-10 mM. At higher concentrations, an appreciably lower incorporation of phosphate into Pr was observed. At optimum concentrations of either MnC12 of MgC12, addition of the other metal ion led only to a decrease of phosphorylation. At optimum metal-ion concentration, the pH optimum was determined to be at pH 7.0-7.5 (Fig. 6). The standard assay was therefore performed at pH 7.2 and 20 mM MgC12. The phosphorylation of phytochrome under standard conditions was used to further characterize the protein-kinase activity (Table 2). The kinase

6.0

I

I

7.0

i

I

8.0

I

pH

Fig. 6. Determination of the pH optima for phosphorylation of A r e n a phytochrome in the presence of 5 mM (o--- 9 or 10-20 mM ( n - - n ) MgClz. Other reaction conditions were as in Fig. 1 ; for determination of radioactivity see Fig. g

required ATP but did not accept guanosine 5'-triphosphate (GTP). That the reaction is indeed a phosphorylation follows from the positive result with [?-32p]ATP and the negative result with [e32p]ATP. Whereas polylysine or cAMP (up to 100 gM) did not stimulate phosphorylation, some stimulation was found with cGMP. About the same stimulation was found with 20 gM Ca 2 + (in addition to 20 mM Mg 2 +). Part of the total activity might be modulated by endogenous calcium and calmodulin. Whereas addition of exogenous calmodulin did not increase the incorporation of phosphate, either the calmodulin inhibitor W-5 or ethylene glycol-bis(/~-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) reduced phosphorylation by 50%. Calcium ions did not increase the reaction in the presence of diolein or phosphatidylserine. Phosphate buffer (20 mM) or glycerol (30%) reduced the reaction by 50%. Neither heparin (9.6mM) nor p-chloromercuribenzenesulfonate (PMBS; up to 3 gM) give rise to appreciable inhibition; the PMBS concentration was sufficient

204

R. Grimm et al. : Phytochrome and protein kinase in oat seedlings

Table 2. Phosphorylation of Arena phytochrome (Pr form) in the presence of several compounds. Standard conditions for phosphorylation in solution were used unless otherwise stated: Tris buffer (20 mM), pH 7.2; 20 mM MgC12 ; 10 gM ATP. The reaction mixtures were then subjected to SDS gel electrophoresis; phytochrome bands were cut out and the radioactivity counted

Compound added

Phosphorylation (standard condition

=1oo%) [7-3~P]ATP [a-3~P]ATP" [~-3~P]GTP" Polylysine (0.1 mg/ml) cAMP (100 pM) cGMP (10 ixM) Ca z+ (20 pM) Calmodulin (i0 pM) Calmodulin inhibitor W-5 b (0.5 mM) EGTA (5 raM) Ca 2+ (500 pM), diolein (16 pg.ml-1) Ca 2+ (500 pM), phosphatidylserine (26 gg. ml-1) Phosphate buffer, pH 7.2 (20 mM) c Glycerol (30%, v/v) Heparin (9.6 nM) PMBS (t 3 pM) Phenylmethylsulfonylfluoride (2 raM) N-tosyl-L-phenylalanine chloromethyl ketone (2 raM) Ne-p-tosyl-L-lysine chloromethyl ketone (2 mM)

100 0 0 100 80 J75 175 100 50 50 90 100 50 50 80 83 35 60 50

Omission of [7-32p]ATP b W- 5 = N-(6-aminohexyl)- 1-naphthalenesulfonamide hydrochoride c Omission of Tris buffer

T z: E "7" ._=

o

128

o

13 L c3_

& N E 13_

. . . .

20

6'0

rain

Fig. 7. Kinetics of phosphorylation of Arena phytochrome. Reaction conditions were as in Fig. 1; for determination of radioactivity see Fig. 5

to modifiy completely one SH-group of phytochrome in the sample (see Eilfeld et al. 1988). Substantial inhibition was found with inhibitors which usually modify serine residues, i.e. phenylmethylsulfonyl fluoride (PMSF), N-tosyl-L-phenylalanine

chloromethyl ketone (TPCK) and Nc~-p-tolyl-L-lysine chloromethyl ketone (TLCK). The kinetics of the phosphorylation reaction under standard conditions are shown in Fig. 7. The reaction was linear only for 5-10 rain under these conditions. The substrate concentration for halfmaximal reaction was determined in the linear part of the kinetics as So.s=5 gM ATP. This means a relatively high affinity of the enzyme for the substrate ATP, comparable to that of other protein kinases (Edelman et al. 1987). Discussion

Phytochrome seems to be tightly associated with one or more protein kinases since even the purest preparations contain at least some kinase activity. This is true for entirely different purification protocols: Wong et al. (1986) purified phytochrome in the Pr form and applied pentyl-agarose chromatography as the main step followed by two hydroxyapatite columns and a polyethylene-glycol precipitation in between (Litts et al. 1983; Lagarias and Mercurio 1985). We used phytochrome purified in the Pfr form by hydroxyapatite chromatography and several washing steps with phosphate buffer (Grimm and Riidiger 1986). Wong et al. (1986) were not able to separate the kinase activity from phytochr0me and concluded therefore that phytochrome itself could be a protein kinase (see also Lagarias et al. 1987). We achieved separation of the kinase activity from phytochrome so that our purest phytochrome preparations are practically devoid of kinase activity (see e.g. Figs. 2, 3). Interestingly, a 50-60-kDa contaminant (see below) has also been found in purified rye phytochrome preparations (Kerscher 1983). This contaminant can only be removed by refined isolation procedures (Ernst and Oesterhelt 1984; Ernst et al. 1987). The phytochrome preparations were not tested, however, for protein-kinase activity in this case. One of the arguments of Lagarias et al. (1987) for phytochrome being a protein kinase is a certain sequence homology with ten known protein kinases. The sequence given by these authors is, however, different from consensus sequences for protein kinases described for 24 (Bairoch and Claverie 1988) or 65 different protein kinases (Hanks et al. 1988). Phytochrome does not contain any of the consensus sequences described in these two papers. This also argues against a protein-kinase role for phytochrome. The kinase activity was localized outside phytochrome, most probably in a 60-kDa protein band,

R. Grimm et al. : Phytochrome and protein kinase in oat seedlings

by two different methods: (i) by incubation of a native gel with labeled ATP and (ii) by application of very low concentrations of ATP in solution. In the first case, only a band with an apparent size of 450 kDa was labeled. Re-electrophoresis with SDS yielded only one labeled 60-kDa band. In the second case, the compound with apparent highest affinity for ATP in the solution of a phytochrome sample was identified as the same 60-kDa protein band (see Fig. 4, lanes a, b). Phytochrome, together with some other minor protein bands, was phosphorylated in this sample only if the concentration of MgC12 was increased to 3 m M or higher. This could mean that the interaction of the kinase with other proteins requires Mg ions (aggregation?) and that only autophosphorylation of the kinase is observed in the absence of such interaction. However, other explanations cannot be excluded since phosphorylation reactions generally require the formation of Mg-ATP complexes as reactive species. It should be mentioned that only 1-2% of total phytochrome is phosphorylated even under optim u m conditions. This is remarkable since oat phytochrome contains a great number of serine and threonine residues (Hershey et al. 1985) as potential phosphorylation sites. Isophytochromes which correspond to known phytochrome genes from oat are present in our preparation in comparable amounts (Grimm et al. 1987) although we cannot exclude the presence of still other isophytochromes in minute amounts. Wong et al. (1986) described a much higher percentage of phytochrome phosphorylation, especially with mammalian proteinkinases A and G. This discrepancy could mean that the protein kinase from oat (presumably the 60-kDa protein) might have a physiological function other than the phosphorylation of phytochrome. Co-purification with phytochrome does not necessarily imply coexistence of phytochrome and the kinase in the same cell compartment. In order to characterize the 60-kDa protein further, we determined its aminoterminal aminoacid sequence. Two conclusions can be drawn from this result: (i) The 60-kDa band is not contaminated with appreciable amounts of other proteins ( > 5%) since the first few Edman steps show only one single sequence in high yield. Contaminating proteins with a blocked amino terminus cannot be excluded, however, by this method. (ii) Since we find a heterogeneity in the ratio of approx. 1 : 1 at steps 5 and 7 and minor heterogeneities at step 9, we must conclude that the 60-kDa protein consists of several isoproteins. The protein band in the native gel at the apparent size of 450-kDa is presum-

205

ably a complex composed of these isoproteins as subunits. Although it is suggestive to assume that one of the isoproteins is the protein kinase, we cannot exclude the possibility that the kinase is a minor component also present in this complex. If so, it must however be tightly associated with the 60-kDa protein because this is phosphorylated at first, even under conditions which exclude phosphorylation of phytochrome (see Fig. 4). We screened a protein-sequence library containing about 14000 protein sequences for the sequence determined here but did not find any related protein. The nature of this protein, the presumed protein kinase, and its precise relationship with phytochrome remain to be elucidated. This work was supported by the Deutsche Forschungsgemeinschaft, Bonn, through grants to E. Schfifer (SFB 206) and to W. Rfidiger. W. R/idiger thanks the Fond der Chemischen Industrie, Frankfurt, FRG, for support. We thank Dr. F. Lottspeich, Martinsried, FRG, for carrying out the amino-acid sequence analysis and Dr. J. Soll, Munich, FRG, for the proteinkinase preparation from pea chloroplast envelope and for the calmodulin antagonist.

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Received 21 October; accepted 14 December 1988