the phosphorylation site of ca2+-dependent protein kinase from alfalfa

Plant Molecular Biology 12: 453-461, 1989 © 1989 Kluwer Academic Publishers. Printed in Belgium 453

The phosphorylation site of Ca 2 +-dependent protein kinase from alfalfa

Zoltan Olah, Laszlo Bogre, Csaba Lehel, Anna Farago ~, Janos Seprodi ~ and Denes Dudits* Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, P.O. Box 521, 6 7 01-Szeged, Hungary (*author for correspondence) 11 st Institute of Biochemistry, Semmelweis University, Medical School, P.O. Box 260, 1444-Budapest 8, Hungary

Received 21 June 1988; accepted in revised form 19 January 1989

Key words: alfalfa, Ca 2 + -dependent protein kinase (CDPK), oligopeptide substrate, peptide competition, protein kinase C, substrate specificity

Abstract

A 50 kDa, calcium-dependent protein kinase (CDPK) was purified about 1000-fold from cultured cells of alfalfa (Medicago varia) on the basis of its histone H 1 phosphorylation activity. The major polypeptide from bovine histone H1 phosphorylated by either animal protein kinase C (PK-C) or by the alfalfa CDPK gave an identical phosphopeptide pattern. The phosphoamino acid determination showed phosphorylation of serine residues in histone H1 by the plant enzyme. Histone-related oligopeptides known to be substrates for animal histone kinases also served as substrates for the alfalfa kinase. Both of the studied peptides (GKKRKRSRKA, AAASFKAKK) inhibited phosphorylation of H1 histones by bovine and alfalfa kinases. The results of competition studies with the nonapeptide (AAASFKAKK), which is a PK-C specific substrate, suggest common features in target recognition between the plant Ca2+-dependent kinase and animal protein kinase C. We also propose that synthetic peptides like AAASFKAKK can. be used as a tool to study substrates of plant kinases in crude cell extracts.

Introduction

In higher plants, as in animals [16], C a 2+ may serve as an intracellular signal transducer for external stimuli, while protein kinases with different Ca 2 + sensitivity are considered as possible effector enzymes [37]. Initially, Ca2+-cal - modulin-activated protein kinases were dis- covered in plants [15,35,36,42]. Later, Ca 2+- -activated protein kinases were detected in the plant cytosol; these kinases need acidic phospho- lipids for activity [10,29,32,43]. Recently a new type of protein kinases has been purified from soybean and from alfalfa using bovine histone H 1 as a substrate. These Ca2+-dependent protein

kinases (CDPK) share common characteristics [3,14].

The bovine histone H1 polypeptide contains several phosphorylation sites for different protein kinases. The use of peptide substrates offers a powerful tool to investigate specificity determinants of various protein kinases. Phosphorylation of the histone H2B-related peptide GKKRKRSRKA by the animal histone kinase II [38,39] and the cyclic nucleotide-dependent protein kinases has been reported [39,41]. For further characterization of the substrate specificity of histone kinase II, the nonapeptide (AAASFKAKK) has been designed [40,41]. It was shown that the enzyme referred to as histone

454

kinase II was identical to the proteolytically activated fragment of the Ca2÷/phospholipid- dependent protein kinase [41]. However, it is worthwhile to mention that the nonapepiide syn- thetized by one of us and a very similar hexa- peptide constructed later by Kondo et al. [20] is considered as the most specific synthetic substrate for PK-C [4,20,39-41].

In our attempts to assess the substrate specificity of CDPK from alfalfa we have used the peptide substrates mentioned above. The results reported in this paper support a conclusion that animal Ca 2 ÷/phospholipid dependent and plant Ca 2 ÷-dependent protein kinases might recognize structurally similar specificity determinants in histone HI.

Materials and methods

Cell materials and culture conditions

An alfalfa (Medicago varia L.cv. Rambler) cell line was established from callus cultures of roots. The cells were grown as suspension cultures in modi- fied Murashige and Skoog medium [27] in the presence of 1 mg/1 2,4-dichlorophenoxyacetic acid (2.4-D) and 200/~g/1 kinetin on a gyrotory shaker (200 rpm) at 28 °C and were subcultured twice a week.

Enzyme purification

All steps wet6 performed at 4 °C. Alfalfa cells (150g) were harvested by filtration through cheese cloth and homogenized by mortar and pestle in 800 ml buffer A: 20 mM Tris-HCl pH 7.4, 50 mM 2-mercaptoethanol and 1 mM phenyl- methylsulfonyl flouride. The homogenate was centrifuged at 100000 x g for 1 hour. The 0-40% saturated (NH4)2SO 4 precipitate of the supernatant was resuspended in 10 ml buffer A and fractionated on a Sephacryl S-300 column (2 x 80 cm) equilibrated with buffer A at 35 ml/h. The column was calibrated with blue dextran, bovine serum albumin, ovalbumin and cyto- chrome C. Fractions (ca. 8 ml) were collected and

40/zl aliquots were tested for protein kinase activity. The active fractions were pooled and subsequently applied to a Q-Sepharose column (2 x 10cm). After washing with buffer B (100 mM NaC1 in buffer A), bound proteins were eluted with a 100-400 mM NaCI gradient in buffer A. The fractions were immediately tested for CDPK activity. Active fractions were concen- trated by (NHa)2SO 4 precipitation (up to 40~o) and redissolved in 10 ml buffer A saturated up to 20~/o by (NH4)2SO 4 (buffer C). This sample was loaded onto a Phenyl-Superose FPLC column (Pharmacia 5/5). The column was washed with buffer C and bound proteins eluted by a decreas- ing gradient made from buffer C and A. Protein contents were determined using bovine serum albumin as standard, according to Lowry et aL [25]. All one-dimensional SDS-PAGE was prepared as described by Laemmli [23]. For two- dimensional separation of proteins the O'Farrell method was used [31].

Protein kinase assay

Samples of 50 #1 were incubated at room temperature. The incubation medium contained: 50 mM KCI, 10 mM MgC12, 25 mM Tris-MES pH 7.4, 20 #M ATP, 0.037 MBq (~-32p)ATP, histone H 1 fraction or the synthetic nonapeptide at 300 #g/ml. The reaction was started by addition of ATP and terminated by the addition of 5 #1 glacial acetic acid. Samples (30/~1) were pipetted onto phosphocellulose paper discs. The discs were washed 3 times for 5 rain with 20 mM NaH2PO4 followed by the same solution containing 1 mM sodium pyrophosphate and 20#M ATP. The radioactivity of the 32p-labelled histone or peptide retained on the phosphocellulose paper was measured.

The peptide substrates (GKKRKRSRKA and AAASFKAKK-amide) were synthesized according to Romhanyi et al. [38,40]. The proteolytically activated protein kinase C fraction from bovine brain was kindly provided by Peter J. Parker (Ludwig Institute for Cancer Research, Imperial Cancer Research Fund, Lincoln's Inn Fields, London, England).

Phosphopeptide mapping

Phosphorylated polypeptides were cut out from SDS-polyacrylamide gels, washed by 25% iso- propanol to remove SDS, and digested with trypsin ( lmg/ml ) overnight at 37 °C. The reactions were terminated by addition of peptide separation buffer: acetic acid, formic acid and H 2 0 ( 15 :5 :80 ) (v/v/v). The digests were lyo- philized and redissolved in the same buffer. Samples were electrophoresed at 600 V on cellulose sheets (Eastman Kodak, No 13255)using 1 ~o acid fuchsin as marker. After drying, chromatography in the second direction was preformed in butanol : pyridine : acetic acid: H20 (32.5 : 25 : 5 : 37.5 v/v/v/v). The dried plates were autoradiographed at - 70 ° C using light enhancer screen. Phosphoaminoacid maps were prepared by the same technique but the digestion was preformed in 6 M HC1.

455

Purification of alfalfa histone H1 fraction

The nuclei were isolated from alfalfa cells origi- nated from the same line used for CDPK purification [13] and the histone H1 [17] fraction was purified according to the method described for other plants. The histone H1 fraction was extracted with 5 ~ (w/v) perchloric acid and precipi- tated by 25 ~ trichloroacetic acid. The pellet was dried from acetone and redissolved in water. This preparation was used as the plant histone HI fraction. The preparation contained three major protein bands referred to as Hla , H l b and H l c according to [ 11 ].

Results

Substrate specifity of the calcium-dependent protein kinase (CDPK) from alfalfa

We have partially purified a soluble C a 2+

-dependent protein kinase from rapidly dividing alfalfa cells as described previously [3]. During

r - .o c~

o

g

t ._= E

21)

QI.

O E t -

6 -

(11

iI1

0 0.05 0.1 02 0.3 0.5 1.0 2.0 3.0 5.0 10

[ s u b s f r a t e ] ,rag m[ -1

Fig. 1. SubstrateconcentrationdependencyofhistoneH1 and synthetic peptide phosphorylation by CPDK isolated from alfalfa. Histone HI ( I ) and oligopeptides (AAASFKAKK, A; GKKRKRSRKA, O) were incubated with enzyme fractions (0.33 #g protein) in the standard incubation media in the presence of I00 #M Ca 2÷ at room temperature for 10 minutes. The measured values were corrected for background measured in the presence of 2 mM EGTA. The average of standard deviations of measurements were signed by vertical bar in the left upper corner.

456

subsequent purification steps bovine histone H1 fraction (Sigma type Ill-S) was used as a substrate. The purification factor relative to the 100000 x g supernatant was 1143 and a specific activity of 12.3 nmol P incorporated into histone H1 per mg protein per min was obtained. The final preparation was enriched in 50 kDa proteins. Using fractions enriched for CDPK activity, we tested different proteins as putative substrates. These studies showed that angiotensin (a tyro- sine-containing polypeptide), bovine serum albumin (with over fifty serines) and casein were not phosphorylated by CDPK (data not shown). In contrast, the histone H 1 fraction and histone- related synthetic peptides were phosphorylated by CDPK in a concentration-dependent manner (Fig. 1). The phosphorylation of both the histone preparation and derived peptides showed non- linear kinetics with substrate inhibition at higher concentrations. The upper limit of enzyme activity for histone was obtained in a lower concentration (0.3mg/ml) than that for the oligopeptides (1 mg/ml for G K K R K R S R K A and 3 mg/ml for AAASFKAKK).

Similar phosphopeptides after phosphorylation of histone HI by plant CDPK and animal PK-C

In an attempt to compare the phosphorylation sites on histone H 1 substrate for plant and animal protein kinases, the tryptic phosphopeptides were analyzed after phosphorylation by CDPK and PK-C. The trypsin digests of the main polypeptide (apparent M r of 31 kDa) from the bovine histone H1 preparation resulted in very similar phosphopeptide patterns (Fig. 2A and B). Further- more, the phosphoamino acid determination revealed that the plant CDPK also phosphorylated serine residues on histone H1 (Fig. 2C, D).

Competition studies with histone-related oligopeptides

As seen in the substrate dependency curves (Fig. 1), both histone H1 and synthetic peptides proved to be inhibitory at higher concentrations. To analyze the substrate-enzyme interactions

Fig. 2. Phosphopeptide map of the major polypeptide of bovine histone H1 phosphorylated previously by CDPK or PK-C. Histone H1 fractions were phosphorylated either by PK-C (part A) or CDPK (part B) and separated in SDS-PAGE. Phos- phorylated polypeptides (apparent Mr 31 kDa) were removed from the PAGE and digested by trypsine. The phosphopeptides were separated horizontally by electrophoresis and then vertically by chromatography on cellulose thin layer and autoradiographed. The phosphoamino determination was carried out after digestion in 6 M HCI and the appropriate standards (panel C) were used to identify the phosphoaminoacid (panel D).

1o0

~ 60

z ~

o ~ 20

8 0

t - C

I

0.6 0.8 1.2 1.6

[Pep t ide ] ,mg rnl "~

C £

Fig. 3. Inhibition of bovine histone H 1 phosphorylation by peptide substrate excess. Histone HI were added into the incubation media in non-limiting concentration (300 #g/ml) with different amount of (AAASFKAKK, • and G K K R K R S R K A &, or together m) peptides in the presence of 100/~M C a 2 +. After separation of the samples by S DS- PAGE the 32p-labelled bands (M r 31 kDa) of histone H1 were cut out from the gel and quantitated by a scintillation counter. All other procedure were performed as described.

457

during phosphorylation, each oligopeptide was applied in excess in the presence of the original substrate. When bovine histone HI (Sigma type III S) was used as a substrate at optimal concentration (0.3 mg/ml, see also Fig. 2), the peptide with basic amino acids near both ends (GKKRKRSRKA) elicited stronger inhibition than the 'PK-C-specific' peptide (AAASFKAKK). Applying the two peptides together resulted in an additive effect (Fig. 3).

The competition assay performed with alfalfa H 1 fractions as protein substrates (Hla = 44 kDa, Hlb = 39 kDa and H l c = 29kDa) also revealed inhibition in the presence of oligopeptides, however the difference between the two peptides was less significant. The Iso calculated from Fig. 4. was approx. 0.82 mM for AAASFKAKK and 0.88 mM for GKKRKRSRKA. Interestingly we found more pronounced differences in inhibitory properties of the two peptides when the soluble fraction of alfalfa cells was used as a competitor. Fig. 5 dem- onstrates that the nonapeptide (AAASFKAKK) considerably inhibited the Ca = + -dependent phosphorylation of plant proteins of 80, 58, 46, 33 and

A A A S F K A K K G K K R K R S R K A C

a b c d e ~ a b c d e Mr M r

x1£~3 kDa

67--

45 - - < 4 4 . . . . . . < 3 9

3 6 - -

29 . . . . . . . . . . . . < 2 9

2 4 - -

2 0 - -

Fig. 4. Inhibition of alfalfa histone H1 phosphorylation by peptide substrate excess. Plant H1 were added into the incubation media in non-limiting concentration (about 300 #g/ml) without (C) or with different amount of oligopeptides (AAASFKAKK or GKKRKRSRKA; a = 0.4; b = 0.8; c = 1.2; d = 2.0 and e = 3.0 mg/ml peptide in the presence of Ca z÷. Incubations and all other procedures were performed as described. Phosphoproteins were separated in 10% SDS-PAGE and autoradiographed. M r of phosphopolypeptides corresponding to H l a, H l b and H l c are signed by arrowheads, downward.

458

Fig. 5. Inhibition of protein phosphorylation in alfalfa curde extract by peptide substrate excess. The cytosol extracted from alfalfa cells were incubated (0.1 mg protein/lane) without (lanes 4, 8, 9) or with different amounts of peptides as competitor (lane 3, 5 = 0.4; lane 2, 6 = 2.0 and lane 1, 7 = 3.0 mg/ml AAASFKAKK or GKKRKRSRKA, respec- tively) in the presence (lane 1-8) or absence (lane 9) of Ca 2 ÷. Incubations and all other procedure were performed as described. Phosphoproteins were separated in 10% SDS PAGE and autoradiographed. Mr of phosphoproteins are signed by arrowheads.

29 kDa. A reduction of phosphorylation signal was also detected at a protein band exhibiting a similar Mr as CDPK. The other oligopeptide ( G K K R K R S R K A ) was less effective. Its inhibitory effect could be detected on phosphoproteins with M r higher than I00 kDa as well as 33 and 29 kDa.

Discussion

In the present paper we have studied the substrate specifity and recognition site of a Ca 2 + -dependent plant protein kinase, using histone H1 and synthetic oligopeptide as substrates. The partially purified alfalfa CDPK [3] enzyme preparation showed several common characteristics with the soybean CDPK [14], such as the Mr, auto- phosphorylation properties, Ca 2 + --dependent mobility shift in SDS-PAGE and substrate specifity. These enzymes did not requi~'e externally added calmodulin for their activity.

H 1 histone in animals is known as a common substrate for a group of different protein kinases which need basic amino acids as specificity determinants. In the histone gene family the H 1 is the most variable, showing tissue and species specificities [5-8,11,45-47]. But contrary to these diversities all histones have some homologous sequences which are conserved during evolu- tion. The first primary structure of a plant histone H 1 has recently been published. It revealed that the sequence surrounding the phenylalanine residue (Table 1) found near to the carboxyl terminus of the globular domain is extremely well conserved [ I, i i ].

Protein kinases are known to recognize the primary sequence of their substrate proteins around the phosphorylated residue [ 19,21,44,48 ]. The animal histone HI contains at least three serine residues (Fig. 2) in the C-terminal part which can be phosphorylated by different animal protein kinases (i.e. sequence No, 97-194, [26]). In animal systems [38,41] the peptide G K K R K R S R K A (derived from histone H2B) has been shown to be an almost equally good substrate for cyclic nucleotide-dependent protein kinases and for the proteolytic fragment of PK-C [40]. Until now there have been no reports of protein kinases other than PK-C which can phosphorylate the peptide A A A S F K A K K [41]. This peptide contains lysines as basic residues and phenylalanine on the C-terminal side of the serine A similar result was obtained from PK-C [20] using a peptide (ASGSFKL) homologous to the histone H 1 consensus (Table 1). The peptide sub-

459

Table 1. Putative consensus phosphorylation domain of CDPK and PK-C in histone HI. H1 °, H5 consensus sequence derived from the amino acid sequences of human HI ° [8], bovine HI ° [34], goose H5 [47], duck H5 [7], and chicken H5 [22]. H1 consensus derived from bovine [24], rabbit [18], boar [6], rat [5], trout [26], Drosophila [28], Xenopus [46] and chicken [22]. H 1 protein sequences by comparing the most frequently used amino acids in the region found near the carboxyl border of the globular domain [11]. The putative recognition site are signed by solid line in the conserved region (dashed line).

Name Sequence Reference

Hi °,H5 G V L K QT K G V GASG SFR L 8 H1 consensus G T L V QT K G T GASG SFK L KK 8 PK-C substrate A S G S F K L 20 PeaHl G K L - - I K - V- - K G SFK L SAAA KK 11 Nonapeptide AAA SFK A KK 40, 41

strates A A A S F K A K K or A S G S F K L both contain this characteristic (SFK). They are suggested to represent the recognition site for PK-C and can also serve as substrates for the plant CDPK. According to the phosphorylation data presented here, the C D P K of alfalfa shares this property with PK-C. A computer search performed on a database of histone sequences did not reveal a SFK sequence in histones other than the above mentioned histone H1 consensus shown in Table i. The mammalian and very likely the plant histone H 1 can be phosphorylated on several serine residues, but only the SFK motif was suggested to be specific for mammalian PK-C. The presence of a SFK sequence in pea histone H1 provides further support for a hypothesis suggesting similar recognition determinants for plant and animal histone phosphorylation.

Interestingly, the alfalfa C D P K followed the same affinity order among the used substrated determined previously [40] for the proteolytic fragment of PK-C (histone kinase II). It is also shown that the tryptic phosphopolypeptides and probably the phosphorylation sites are similar, either the animal PK-C or the C D P K from alfalfa were used to phosphorylate the major component of histone H1 fraction (Fig. 3). In addition to these similarities, the alfalfa C D P K and animal PK-C differ in their ability to phosphorylate core histones and a 16 kDa nuclear protein (L. Bogre and D. Dudits, unpublished).

On the basis of competition studies, one can conclude that the synthetic peptides compete with

phosphorylation sites in substrate proteins. The competition assay provided different patterns in the case of PK-C 'specific' and 'non-specific' peptides when various substrated were compared. Using the plant C D P K we found that the G K K R K R S K A peptide was more efficient than A A A S F K A K K in inhibiting bovine histone H1 phosphorylation. This observation is in agree- ment with the results of experiments with animal histone kinase II [40]. The efficiency order might reflect the differences in Km values which were 0 .02mM for histone H1, 0 .11mM for G K K R K R S K A and 0.22 mM for A A A S F K A K K when the mammalian enzyme was used [40]. The most obvious difference between the two oligopeptides was detected in inhibition of protein phosphorylation in the cell extract.

In animal cells, the second messenger-related protein kinases are known to be involved in trans- membrane signalling and intracellular signal transmission to the cell nucleus. One of the families of these protein phosphorylating enzymes are the Ca2+/phospholopid-dependent protein kinases [2,30,33]. Considering some characteristic features of the kinase described in this report, we would like to suggest similarities with PK-C, particulary for target recognition of animal and plant histone H I as substrates. Recently it has been shown that a polyclonal antibody raised against the polypeptide sequence (280-292) of bovine brain PK-C could also recognize protein bands of similar M r from a plant extract [9].

In summary, although it is difficult to determine

460

the extent of homology of animal Ca 2 +/phospholipid dependent [2,16,30] and plant Ca 2+ -dependent protein kinases [ 14,15,43], it is clear that the two enzymes show significant similarities in substrate specificity and phosphorylation sites. The substrate specificity of the plant and animal Ca2+-dependent protein kinases presumably relies on the aminoacid residues surrounding the phosphorylatable serine and/or on the similarity of substrate binding sites. Furthermore we suggest that the PK-C 'specific' peptide (AAASFKAKK) can be used as a competitor to characterize the intrinsic properties of CDPK in crude cellular extracts.

References

1. Allan J, Harman PG, Crane-Robinson C, Aviles, FX: The structure ofhistone H 1 and its location in chromatin. Nature 288:675-679 (1980).

2. Ashendel CL: The phorboester receptor: a phospholipid-regulated protein kinase. Biochim Biophys Acta 822:219-242 (1985).

3. Bogre L, Olah Z, Dudits D: CaZ+-dependent protein kinase from alfalfa (Medicago varia): partial purification and autophosphorilation. Plant Science 85:135-144 (1988).

4. Buday L, Seprodi J, Farkas Gy, Meszaros Gy, Romhanyi T, Banhegyi G, Mandl J, Antoni F, Farago A: Proteolytic activation of protein kinase C in the extracts of cells treated for a short time with phorbol ester. FEBS Lett 223:15-19 (1987).

5. Cole KD, York RG, Kistler WS: The amino acid sequence of boar Hit , a testis-specific HI histone variant. J Biol Chem 259:13695-13702 (1984).

6. Cole KD, YorkRG, Kistler WS: Sequence ofthe amino- terminal half of the rat testis-specific histone variant H 1 t. Biochim Biophys Acta 869:223-229 (1986).

7. Doenecke D, Tonjes R: Conserved dyad symmetry structures at the 3' end of H5 histone genes. J Moi Biol 178:121-135 (1984).

8. Doenecke D, Tonjes R: Differential distribution oflysine and arginine residues in the closely related histones H 1 ° and H5. Analysis of a human HI ° gene. J Mol Biol 187: 461-464 (1986).

9. Elliott DC, Kokke YS: Cross-reaction of a plant protein kinase with antiserum raised against a sequence from bovine brain protein kinase C regulatory sub-unit. Biochem Biophys Res Commun 145:1043-1047 (1987).

10. Elliot DC, Skinner JD: Calcium-dependent, phospholipid activated protein kinase in plants. Phytochemistry 2:39-44 (1986).

11. Gantt JS, Key JL: Molecular cloning of pea H 1 histone eDNA. Eur J Biochem 166:119-125 (1987).

12. Graziana A, Fosset M, Ranjeva R, Hetherington AM, Lazdunski M: Ca 2 ÷ channel inhibitors that bind to plant cell membranes block Ca 2÷ entry into protoplasts. Biochemistry 27:764-768 (1988).

13. Hadlaczky Gy, Bisztray Gy, Praznovszky T, Dudits D: Mass isolation of plant chromosomes and nuclei. Planta 157:278-285 (1983).

14. Marmon AC, Putman-Evans C, Cormier M J: A calcium- dependent but calmodulin-independent protein kinase from soybean. Plant Physiol 83:830-837 (1987).

15. Hetherington A, Trewavas A: Calcium-dependent protein kinase in pea shoot membranes. FEBS Lett 145: 67-71 (1982).

16. Hunter T: Thousand and one protein kinases. Cell 50: 823-829 (1987).

17. Jones EW: The isolation and purification ofhistones. In: Prescott DM (ed) Methods of Cell Biology, Vol. 16, pp. 183-203. Academic Press, New York/London (1977).

18. Jones GMT, Rail SC, Cole RL: Extension of the amino acid sequence of a lysine-rich histone. J Biol Chem 249: 2548-2553 (1974).

19. Kemp BE, Graves DL, Benjamini E, Krebs EG: Role of multiple basic residues in determining the substrate specificity of cyclic AMP-dependent protein kinase. J Biol Chem 252:4888-4894 (1977).

20. Kondo H, Baba Y, Takaki K, Kondo K, Kagamiyama H: Phosphorylation by protein kinase C of a synthetic hepta- peptide bearing a lysine residue on the C-terminal side of serine. Biochem Biophys Res Commun 142:155-161 (1987).

21. Krebs EG, Beavo JA: Phosphorylation-dephosphoryla- tion of enzymes. Ann Rev Biochem 48:923-959 (1979).

22. Krieg PA, Robins AJ, Colman A, Wells JRE: Chicken histone H5 mRNA: the polyadenylated RNA lacks the conserved histone 3' terminator sequence. Nucleic Acids Res 10:6777-6785 (1982).

23. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 (1970).

24. Liao LW, Cole RD: the amino acid sequence of residues 1-104 of CTL-1, a bovine H1 histone. J Biol Chem 256: 3024-3029 (1981).

25. Lowry OH, Rosebrough NS, Farr AL, Randall RJ: Pro- tein measurement with the Folin phenol reagent. J Biol Chem 193:265-275 (1959).

26. Macleod AR, Wong NCW, Dixon GH: The amino-acid sequence of trout-testis historic HI. Eur J Biochem 78: 281-291 (1977).

27. Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473-497 (1962).

28. Murphy TJ, Blumenfeld M: Nucleotide sequence of a Drosophila melanogaster H1 histone gene. Nucleic Acids Res 14:5563 (1986).

29. Muto S, Shimogawara K: Calcium- and phospholipid- dependent phosphorylation ofribulose- 1,5-bisphosphate carboxylase small subunit by chloroplast envelope- bound protein kinase in situ. FEBS Lett 193:88-92 (1985).

30. Nishizuka Y: The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature (London) 308:693-698 (1984).

31. O'Farrell PH: High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007-4021 (1975).

32. Olah Z, Kiss Z: Occurrence of lipid and phorbol ester activated protein kinase in wheat cells. FEBS Lett 195: 33-37 (1986).

33. Parker PJ, Stabel S, Waterfild MD: Purification to homogenity of protein kinase C from bovine brain: identity with the phorbol ester receptor. EMBO J 3: 953-959 (1984).

34. Pehrson JR, Cole RD: Bovine Hl ° histone subfractions contain an invariant sequence which matches histone H5 rather than H1. Biochemistry 20:2298-2301 (1981).

35. Polya GM, Davies JR: Resolution of Ca 2 ÷-calmOdulin-

activated protein kinase from wheat germ. FEBS Lett 150:167-171 (1982).

36. Polya GM, Micucci V: Partial purification and characterization of a second calmodulin-activated Ca 2+ -dependent protein kinase from wheat germ. Biochim Biophys Acta 785:68-74 (1984).

37. Poovaiah AS, Reddy N, McFadden JJ: Calcium messenger system: role of protein phosphorylation and inositol bisphospholipids. Physiol Plant 69:569-573 (1987).

38. Romhanyi T, Seprodi J, Antoni F, Meszaros G, Farago A: The site of histone H2b phosphorylated by a cyclic nucleotide independent histone kinase. Biochim Biophys Acta 701:57-62 (1982).

39. Romhanyi T, Seprodi J, Meszaros Gy, Antoni F, Farago A: The intrinsic substrate specificity of cyclic nucleotide- independent protein (histone) kinase. FEBS Lett 144: 223-225 (1982).

40. Romhanyi T, Seprodi J, Antoni F, Meszaros Gy, Farago

461

A: Specific substrate for histone kinase II: a synthetic nonapeptide. Biochim Biophys Acta 827:144-149 (1985).

41. Romhanyi T, Seprodi J, Antoni F, Meszaros Gy, Buday L, Farago A: The assay of the activity of protein kinase C with the synthetic oligopeptide substrate designed for histone kinase II. Biochim Biophys Acta 888:325-331 (1986).

42. Salimath MJ, Marme D: Protein phosphorylation and its regulation by calcium and calmodulin in membrane fractions from zucchini hypocotyls. Planta 158:560-568 (1983).

43. Schafer A, Bygrave F, Matzenauer S, Marme D: Identification of a calcium- and phospholipid-dependent protein kinase in plant tissue. FEBS Lett 187:25-28 (1985).

44. Small D, Chou PY, Fasman D: Occurrence of phosphorylated residues in predicted -turns: implications for -turn participation in control mechanisms. Biochem Biophys Res Commun 79:341-346 (1977).

45. Sugarman BJ, Dodgson JB, Engel JD: Genomic organi- zation, DNA sequence, and expression of chicken embryonic histone genes. J Biol Chem 258:9005-9016 (1983).

46. Turner PC, Aldridge TC, Woodland HR, Old RW: Nucleotide sequences of i l l histone genes from Xenopus laevis. A recently diverged pair of H1 genes and an unusual H1 pseudogene. Nucleic Acids Res 11: 4093-4107 (1983).

47. Yaguchy M, Roy C, Seligy VL: Complete amino acid sequence of goose erythrocyte H5 histone and the homology between HI and H5 histones. Biochem Biophys Res Commun 90:1400-1406 (1979).

48. Zetterqvist O, Ragnarsson U: Humble E, Berglund L, Engstrom L: The minimum substrate of cyclic AMP- stimulated protein kinase, as studied by synthetic peptides representing the phosphorylatable site of piruvate kinase (type L) of rat liver. Biochem Biophys Res Com- mun 70:696-703 (1976).

the phosphorylation site of ca2+-dependent protein kinase from alfalfa

Documents