Amino acid sequence of rat thymus histone H2B and identification of the in vitro phosphorylation sites

BIOCHIMIE, 1979, 61, 61-69.

Amino acid sequence of rat thymus histone H2B and identification of the in itro phosphorylation sites. Arlette MARTINAGE, Paul MANGEAT *, Pierre SAUTII~.RE <>, Ouy MARCHIS-~OUR'EN * and G6rard BISERTE. (29-8-1978).

Unit~ 124 I N S E R M el Institul de R e c h e r c h e s sur le Cancer de Lille, B.P. n ° 3567, 59020 Lille Cddex and * lnstitut de Chimie Biologique, Universit~ d'Aix-Marseille, Place Victor-Hugo, 13331 Marseille C~dex 03, France.

R6sum~.

Summary.

La s 6 q u e n c e en a c i d e s a m i n 6 s de l'histone de t h y m u s de rat, o b t e n u e ~ r 6 t a t tr~s p u t p a r 61ectrophor~se pr6parative, a 6t6 d6termin6e. Cette s 6 q u e n c e est identique ~ celle de l'histone H2B de t h y m u s de v e a u . La phosphor¥1ation in vitro de l'histone de rat a v e c une prot6ine kinase activ6e p a r I'AMP cyclique isol6e du p a n c r 6 a s de rat, conduit & l'identification de q u a t r e sites de p h o s p h o r y l a t i o n : deux sites majeurs, non sp6cifiques, sur les r6sidus de s6rine 32 et 36, et deux sites mineurs, sp6cifiques de la prot6ine k i n a s e de rat, sur les r6sidus de s6rine 87 et 91.

The a m i n o acid s e q u e n c e of rat t h y m u s histone obtained in h i q h l y purified form b y preparative electrophoresis, w a s determined. This s e q u e n c e is identical to the s e q u e n c e of calf t h y m u s histone H2B. The in vitro p h o s p h o r y l a tion of the rat histone with a cyclic AMP-dependent protein kinase isolated from rat p a n c r e a s led to the identification of four sites of phosphorylation : two m a j o r ones, at serine residues 32 a n d 36, a n d two minor ones, specific of the rat protein kinase, at serine residues 87 a n d 91.

Introduction.

Two ,major sites of in vitro phosphorylation from si.lkworm [5] ov from bovine cerebellum [6] have been localized at serin, e 32 and serine 36 in calf histone H2B.

The primary structure of histone H2B has been determined in three species, calf [1], trout [21 and more recently sea-urchin [3, 4] and a comparative s.tudy of the sequences has shown that most of the observed changes occurred in the N4erminal ha,lf of the molecule. The N-terminal sequence of the histone H2B is highly ba~i.c and ,is probably a privileged site of electroslatic interactions with the phosphate groups of the DNA. The r~gulation of these interactions is ensured by a system of phosphorylation-dephosphoryl~ation.

The abbreviations used are : Cyclic AMP, adenosine 3' :5'-monophosphate ; TPCK, T.-(l-tosylamido-2-phenyl) ethyl chloromethyl ketone. ~> To whom all correspondence should be addressed.

.From these results, the enzyme does not seem to have any species specific~iy. In order to ascertain this lack of specificity we have prepared the cyclic AMP-dependent protein kin3se from rat pancreas and vc,e have used the rat thymus histone H2B as substraie for our phosphorylation assays in vitro. The amin,o a.cid sequence of the rat histone H2B yeas deduced from the structural data provided by the tryptic peptides separated by ion exchange column chromatography. This methodology set up for separalion of ~nlabelled peptides was then applied to that of [~P] labelled p,eptides. Four sites of phos.phorylation were identified in rat thymus histone H2B : two major sites, at serine residues 32 and 36, two minor at

A. Martinage

62

s e r i n e r e s i d u e s 87 a n d 91. T h e t w o m i n o r s i t e s a p p e a r to be s p e c i f i c f o r t h e r a t p a n c r e a s p r o t e i n kin,use.

Materials and Methods. T h y m u s g l a n d s excised f r o m t w o m o n t h old W4star Rats, w e r e f r o z e n i m m e d i a t e l y on d r y ice a n d kept at - - 3 0 ° C u n t i l use.

Isolation of histone H2B. T h e F2b fracti~on wa,s ~ b t a i n e d f r o m r a t t h y m u s ehvom a t i n b y t h e m e t h o d described b y J~h.ns [7]. T h i s fraetion~ c o n t a i n e d h i s t o n e It2B b u t ,also h i s t o n e s H3 a n d H2'A. t~ighly purified hi.stone H~B was. ~sol,ated b y p r e p a r a t i v e ,eleetrophoresis a t pH %7, in 2,.5 M urea, on po.lyaerylamide slab gel aeeordin.g to M a r t i n a g e et

at. [8]. P u r i t y of the preparatio.n w a s a s s e s s e d by an,alytical gel e l e c t r o F h o r e s i s a s described l~reviously [8].

A m i n o acid analgses. H'istone s a m p ~ s w e r e h y ~ r o l y z e d in vacuo a t l l 0 ° C f o r 24, 48 a n d 72~ b in~ 6 M HG1. O,ne drop of 1 per cent p h e n o l wa.s added to p r e v e n t exces s~ive d e g r a d a t i t m of t y r o s inc. A m i n o acid *analyses were p e r f o r m e d on a Beekm~a~ M n i t i c h r o m am.i~no acid an.alyser e q u i p p e d w i t h a n I~nfotron.ics Integrato,r model CRS-2:10.

End group d e t e r m i n a t i o n . The a m i n o t e r m i n a l group of b i s t o n e I-I2B w a s determ,lned b y da,nsylation a c c o r d i n g Vo t h e m e t h o d of Gros a n d L a b o u e s s e [9]. For t h e carboxy-termin~ai s e q u e n c e d e t e r m i n a t i o n , t h e histo,ne H~B was, h y d r a l y z e d in. 0.2 IV[;N - e t h y l m o r p h ~ 4 n e b~ffer, pH 8.'5 a t 4 0 ° ~ w i t h c a r b o x y p e p t i d a s e s B ~md A. A l i q u o t s were t a k e n off at t i m e points, freeze dried a n d a n a l y s e d on t h e a m i n o acid a~nalyser.

Tryptic hydrolysis. T h e p r o t e i n w a s b y d r o l y z e d for 2 h o u r s at pH 8.0 a n d 37°{2 in 0.1 M a m m o n i u m b i c a r b o n a t e w i t h TPCKt r e a t e d t r y p s i n ( W o r t h i n g t o n ) , u s i n g an. e n z y m e to s u b s t r a t e ratio of 1/50. H y d r o l y s i s w a s stopped by l o w e r i n g t h e pH to 3.5 w i t h f o r m i c acid. E r a e t i o n a t i o ~ ¢ff t h e t r y p t i c p e p t i d e s w a s p e r f o r m e d on G h r o m o ~ e a d s P c o l u m n (Tech.nieon Corporation) w i t h p y r i d i n e - f o r m a t e a n d p y r i d i n e - a c e t a t e b u f f e r s u s e d i n a g r a d i e n t s y s t e m f r o m 0.1 M in p y r i d i n e

a n d coll. at pH 2.9 to 2.0 M in pyrid~ne at pH 5.0 [10], F ~ r t h e r p u r i f i c a t i o n of t h e p e p t i d e s w a s achieved a;s described p r e v i o u s l y [11]. T h e s e q u e n c e of t h e peptides w a s estabti,shed by E d m a n d e g r a d a t i o n w i t h direct identification of phen3dthiohydantoi~n a m i n o acids o n silica gel t h i n d . a y e r m i c r o c h r o m a t o g r a p h y as described by Gohen-Sola~l a n d B e r n a r d [12]. l ~ h e n y l t h i o h y d a n t o i n deriwatives nf h i s ti~ine a u d a r g i n i n e w e r e identified b y h i g h voltage e l e c t r o p h ~ r c s i s un, W h a t m a , n 3 MM p a p e r fallowed b y specific s t a i n i n g w i t h P a u l y r e a g e n t a n d phen,a,nt h r e n e q u i n o n e r ~ a g e n t [13] respectively.

Protein-kinase extraction and purification. T h e procedure of p u r i f i c a t i o n w h i c h l e a d s to t h e i s o l a t i o n of t h e c a t a l y t i c s,ubunit of t h e cyclic AMPd e p e n d e n t proteir~ k i n a s e , is a slig,ht m o d i f i c a t i o n of t h e procedure, previo,usly described [14]. Pancreas~ f r o m 15 mal,e W'istar R a t s ~w e i g h i n g 25,0 g, were h o m o g e n i z e d in, 0.3 M sucrose c o n t a i n i n g 1 mM b e n z a m i d i n e a n d 6 m M ~ - m e r c a p t o e t b a n o l . The h o m o gen,ate w a s c e n t r i f u g e d for 1.5 h a t 60.000 r p m in a 65 B.eckman r o t o r a n d the s u p e r n a t a n t (S~) collected. T h e pellet w a s res~,spended in a pH 7.8 m e d i u m c o n t a i n i n g 50 m M NaC1, 125 mM NaHCO~, 1 m M b e n z a m i d i n e , 6 m M ,~-meroaptoe~l~anol. The s u s p e n s i o n w a s c e n t r i f u g e d for 45. m i n in. th,e s a m e conditions' a s above. T h e s u p e r n a t . a n t (S~) w a s collected. S~ a n d S~ were pooled a n d (NH4)~SO~ was added up to 2.4 M. final ct>nc e n t r a t i o n . After c e n t r i f u g a t i o n t h e s u p e r n a t a n t w a s disca~'ded a n d t h e pel!ict w a s dissolved i n 30 ml of 50 mM s o d i u m p h o s p h h t e pH 6.8 c o n t a i n i n g 1 mM benz a m i d i n e , 2 m M EDTA a n d 6 mM ~ - m e r c a p t o e t h a n o l . T h i s s o l u t i o n w a s filtered t h r o u g h a 5 × 100 cm G 75-8ephadex e61umn equ~ilibrated w i t h t h e s a m e b u f fer. T h e 1-1'.5 Vo eluted f r a c t i o n w a s pooled a n d loaded ont, o a 1.5 X 3~) cm DEAE-Sepbaros~ c o l n m n e q u i l i b r a t e d w i t h t h e s a m e buffer. After w a s h i n g t h e c o l u m n w i t h t h e e q n f l i b r a t i o n buffer t h e p r o t e i n k i n a s e vr~s eluted b y t h e s a m e buffer except t h a t t h e p h o s p h a t e c o n c e n t r a t i o n w a s 45 mM a.nd t h a t 10 ~M cyclic AMP w a s present. The f r a c t i o n s c o n t a i n i n g the e n z y m e w e r e pooled a n d d i l u t e d twice w i t h w a t e r a n d loaded onto a 1.5 × 30 em CM-Sepha.rose c o l u m n e q u i l i b r a t e d w i t h 2.5 mM s o d i u m p h o s p h a t e buffer (pH 6~.8) c o n t a i n i n g 1 m M b e n ~ a m i d i n e a n d 6 mM ~ - m e r c a p t o e t h a n ~ l~. The e n z y m e was eluted b y ~ .linear g r a d i e n t of NaGl f r o m 0 to 0.3 M u s i n g 209 ml of sta.rting buffer a n d $00 ml of final buffer.

P h o s p h o r y l a t i o n conditions. About 2~) m g of h i s t o n e were i.ucubated for 24 h at 30 ° (the h i s t o n e final c o n c e n t r a t i o n was, 0.5 m g / m l ) , in t h e p r e s e n c e of 0.4 mM ~t~2P ATP (75 ~Ci), 30 mM Tris HCA pH 7.8 a n d 12. mM MgGl_o. The e n z y m e conc e n t r a t i o n w a s ~1-1.5 ~ g / m l . The i n c u b a t e w a s dialyzed a g a i n s t w a t e r (6 1) f o r 24 h a n d iyophHized.

Fro. 1. - - Peplide maps on W h a t m a n 3 MM paper of the tryplic digests of calf and rat H2B. Ghrom;atography (C) w a s r u n for 18 h o u r s in the s o l v e n t s y s t e m : 1 - b u t a n o l - p y r i d i n c - a c e t i c a c i d - w a t e r (15:10:3:12) (v/v% E l e c t r o p h o r e s i s (E) w a s r u n for 90 m i n u t e s at 2 200 volts in 'a p y r i d i n e - a c e t a t e buffer pH 3.6. Maps were stain.ed w i t h n i n h y d r i n - c a d m i u m r e a g e n t [15].

BIOCHIMIE, 1979, 61, n ° 1.

i

~'i ~ii~ii~~~!//i,~'~/i,,~!:~ i~!!~!iii!~!iii!!i~!ii! :'¸~:' ~~i~/:~

/~iI :

;i:¸::

~ ~;~

,~

ARG Jr

A. Martinage and coll.

64

Tryptic hydrolysis and location of radioactioe peptides. 2,1 mg of p r o t e i n labelled w i t h [32p] were dissolved i~ 2.5 ml of 0.1 M N - m e t h y l m o r p h o l i ~ e , pH 8.0 a~nd h y d r o l y z e d for 2 hours, a t 37°C w i t h TPCK-treated t r y p s i n u s i n g a n enzyme to s u b s t r a t e r a t i o of 1/50'. &n aliquo~ of the hydro~ysate wa~s t~ken for peptide m a p p i n g on W h a t m , a n 3 MM paper. Before s t a i n i n g w i t h t h e nin~hydrine-cadmium r e a g e n t [15], t h e radioactive peptides were located o n t h e m a p b y radioa u t o g r a p h y , u s i n g Kodirex film 45 × 45 cm (Kodak) wi~h a 4& h o u r s exposure. The trTptie hyd~oly.s~te ~a.s fraetion, ated b y ion exchange c h r o m a t o g r a p h y as described above. E l u t i o n of the peptides was m o n i t o r e d automatioa,l,ly b y nin~ hydrin, reaction. The r a d i o a c t i v i t y w a s t h e ~ m e a s u r e d w i t h a liquid s c i n t i l l a t i o n c o u n t e r (Nuclear Chicago) on a n a l i q u o t of each peptidic fracticm.

Results. R a t t h y m u s h~istoine H2~B, i n h i g h l y p u r i f i e d f o r m , w,as o b t a i n e d fro,m f r a c t i o n F2b o f J!ohns b y preparative electrophoresis, using a polyacryla m i d e s l a b gel, w i t h a y i e l d of a b o u t 45 p e r c e n t . Histone H2B was characterized by its electroph,oretic mobility in an analytical polyacrylami,de geI a t p H 2.7 a n d b y its amin,o a c i d c o m p o s i t i o n w h i c h is p r e s e n t e d i n ~ahle I. T h e c o m p o s i t i o , n of t h i s h i s i o n e is v e r y s i m i l a r to t h a i of t h e c a l f t h y m u s h o m o l o g o u s h i s t o n e . T h e a m i n o - t e r m i n a l groaxp w a s f o u n d to h e p r o line, after dansylation.

TABLE I.

Amino acid composition of the H2B historic [rom rat thymus. Moles per cent

Calculated residues

Amino acids

Aspartic acid Threonine (*) Serine(*) Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrostne Phenylalanine Histidine Lysine Arginine

Rat thymus

Calf thymus

5.14 6.05 11 08 7.99 4.61 6.08 10.24 7.14 1.37 4.71 5.46 3.61 1.38 2.57 15.90 6.63

4.9 6.1 10.7 7.6 4.8 5.5 10.1 7.0 1.5 4.9 4.9 3.8 1.6 2.2 16.6 6.2

Rat thymus 6.4 7.6 13.8 10.0 5.8 7.6 12.8 8.9 1.7 5.9 6.8 4.5 1.7 3.2 19.9 8.3

(6) (8) (14) (10) (6) (7) ('*) (13) (9) (2) (6) (6)(") (5) (2) (3) (20) (8)

Calf thymus 6 8 14 10 6 7 13 9 2 6 6 5 2 3 20 8

Values, expressed as moles per cent of all a m i n o acids are average of 9 d e t e r m i n a t i o n s w i t h 24, 48 and 72 h o u r s - h y d r o l y s a t e s . A m i n o acid composition of the H2B h i s t o n e from calf t h y m u s [1] is given for comparison. (*) Values for t h r e o n i n e a n d serine are o b t a i n e d by l i n e a r e x t r a p o l a t i o n to zero time of hydrolysis. (**) Values deduced f r o m the sequence study.

Amiuo a.cid compositi,ou of each p h o s p h o p e p t i d e was d e t e r m i n e d a f l e r h y d r o l y s i s in 6 N H~C1 for 24 h a t ll,0°C u n d e r vacuum. I~n the,se oondi¢i,o~s, t h e r a t e of decompos4tio~ of O - p h o s p h o a m i n o acids was complete [16].

The carhoxy-term~na'l sequence Ser-Lys was d e d u c e d f r o m t h e s t u d y of t h e h y d r o l y s i s of t h e p r a t e i n w i t h c a r b o x y p e p t i d a s e s B a n d A.

The p h o s p h o r y l a t e d sites in h i s t o n e s I ~ B were identified by sequencing t h e phosphop~ptides b y E d m a n d e g r a d a t i o n a n d m e a s u r i n g the r a d i o a c t i v i t y a f t e r each cycle involving the e l i m i n a t i o n of a serine residue.

The insoluble material which precipitated during the tryptic hydrolysis, was removed by cent r i f u g a t i o n . Th4s m a t e r i a l , s o l u b l e i n 30 p e r c e n t a c e t i c a c i d c o r r e s p o n d s to t h e t r y p i i c i n s o l u b l e

BIOCHIMIE, 1979, 61, n ° 1.

Sequence of rat histone H2B and phosphorglation sites.

65

T4 T18

A 570nrn ' N H 3

T20 free T14 Arg2 1Tt i

nfree ~L

1.0

/

~2

II

II,

.7

T6

~

T

T5

TIO

1 ~ / t

1 2 L, 275

3 456

7 891011 12 13 I 550

14 1516 17 I 825

I 1100

T3a

T7

I"

1.0

TIOa

19

0

T

T15

J_

T.

Effluent(ml)

2o

21

~

I 1650

I ~375

2-3"-

I 1925

..--

I 2200

Effluent[m!)

Elulion diagram of soluble trgplic peptides of hislone H2B from rat thgmus, fractionated on Chromobeads P column. P~ptides were eluted w i t h p y r i d i n e - f o r m a t e a n d p y r i d i n e - a e e t a t e buffers FIG. 2.

-

-

u s e d i n a g r a d i e n t of c o n c e n t r a t i o n a n d pH. P e p t i d e s were d e s i g n a t e d bv the s y m b o l T a n d n u m b e r e d a ccord.ing to t h e i r pos.ition in t h e a m i n o acid s e q u e n c e of the protein. " IO 20 P ro-Glu-Pro-Nla-Lys-S er-Al a-Pro-Nla-Pro-Lys-Lys-Gly-Ser-Lys-Lys-Nla-Val-Thr-Lys-Nla-Gln-Lys,~

t

T-2 ~

4-- T - 3 - - - ~

i"

T-3a

~

T-4

~

~

q

•

T-5 --I) T-5-a

30 ,' 40 Lys-Asp- Gly-Lys-Lys-Arg-Lys-Arg- Ser-Arg-Lys-Glu-Ser-Tyr-Ser -Val-Tyr-Va l-Tyr -gys-Val-Leu-Lys•

T-6a l, 41--- T-6

~ 41-T-7-4p 4--T-8--~ ~"---T-9-----P 4 ), •

,

T-1 0 T-lOa

-I. •

T-11-----~

50 60 (Gln, Val, His, Pro, Asp, Thr) Gly-I le-Ser-Ser-Lys-Ala-Met-Gly-I le-Met-Asn-Ser-Phe-Val-Asn-Asp (I le, (f

T-12 T h - A ~ P T ~ L T-12

T-12 Th-B------) ~ 4

T-13

70 80 90 Phe•G•x)Arg-••e-A•a-G•y-G•u-A•a-Ser-Arg-Leu-A•a-His-Tyr-Asn-Lys-Arg-Ser-Thr-••e-Thr-Ser-Arg-

)~

T-14

~4

T-15,

b

4

T-16

100 110 Glu-I•e-Gln-Thr-Ala-Va•-Ar•-Leu-Leu-Leu-Pr•-Gly-Glu-Leu-A•a-L•s-His-A•a-Val-Ser-Glu-•ly-Thr~

T-17

~

~

T-18

)

4

T-19

120 125 Lys-AI a-Val-Thr-Lys-Tyr-Thr- Ser-Ser-Lys (OH) FIG. 3. -- A m i n o acid sequence of hlstone H2B from rat thymus. T h e p,arti,al seq,uen,ce of t'he p e p t i d e T - I ~ w a s d e d u c e d f r o m t h e s t r u c t u r a l d a t a p r o v i d e d b y t h e p e p t i d e s T-12'-Th-A a n d T-I~-Th-B~. T h e s e p e p i i d e s w e r e o b t a i u d e b y c l e a v a g e of t h e p e p t i d e T-19 w i t h t h e r m o l y s i n . The s i t e o f c l e a v a g e ,is, i n d i e a : t e d b y a n a r r o w a n d l e t t e r e d b y Th. M e t h o d s u s e d f o r d e t e r m i n a t i o n of t h e ~ e q u e n c e of p e p t i d e s 'are i a d i e a t e d a s f o l l o w s : m a n u a l E d m a n d e g r a d a t i o n -+ ; Ca r b o x y - p e p t i d a s e s A a n d B h y d , r o l y si~s ~--,

BIOCHIMIE, 1 9 7 9 ,

61, n ° 1.

A. Martinage and coll.

66

core coming from the hydrophohJc sequence l o c a t e d in t h e m e d i a n p a r t of t h e p r o t e i n ,

T h e e l a t i o n d i a g r a m of t h e s o l u b l e t r y p t i c p e p t i d e s is s h o w n in figure 2. Most of t h e p e p t i d e s

TABLE II.

Amino acid composition o[ the lryptic peptides of rat thymus histone H2B. Amino acids

T-t

Aspartie acid Threonine Serine Glutamic acid Proline Glyeine Alanine Valine Methionine Isoleueine Leueine Tyrosine Phenylalanine Lysine Histidine Arginine Number of residues Yield (per cent)

T-2

T-3

T-4 T-20

T-3a

T-5

T-5a

'1"-6

T-7 T-8

T~a

T-9

T-t0

T-t0a

1.o (1) 1.2 (1) 1.0 (t)

0.8 (1) 1.4 (2) 1.5 (2) 1.0 (l) 1.3 (1)

0.8 (l) 0.9 (1) 1.2 (1) 1.1 (1) 1.0 (1) 0.9 (1) 1.8 (2) 2.0 (2) 1.3 (t) t . 4 (t) 1.1 (1) 1.3 (1) 1.1 (1) 0.9 (1) 1.1 (1) 1.0 (1) 1.9 (2) 1.0 (t)

1.9 (2) t . 8 (2)

2.7 (3) 2.4 (3) 1 0 (1) 1.0 (1) 1.0 (1) 2.0 (2) 1.0 (I) 1.O(1) 2.0 (2) 1 . 0 ( 1 ) 2.0 (2) 1.0(1) 1 . 0 ( 1 ) 1.1 (1) 2.2 (2)

1.0 (1) 1.o (1) 5 10.8

6 17 3

3 2.0

4 13.0

4 28.0

3 12.1

4 2.6

3 4.3

4 2.6

2 6.5

3 8.6

9 6.0

I0 2.8

TABLE 11 ( c o n t i n u e d ) .

Amino acid composition of the tryptic peptides o[ rat thymus histone HflB. Amino acids Aspartie acid Threonine Serine Glutamie acid Proline Glyeine Alanine Valine Metilionine Isoleucine Leueinc Tyrosine Phenylalanine Lysine Histidine Arginine Number of residues Yield (per cent)

T-II

T-t2 0.9 0.9 1.6 1.0 I .0 1.0

0.9 (1)

(1) (1) (2) (1) (1) (1)

0.9 (1) 0.9 (1)

T-t3

T-t~

3.0 (3)

T-t6

T-t7

1.8 (2) 1.9 (2)

l 0 (1)

T-t8

T-t9

1"-2t

1.0 (1) 1.2 (1) 1.0 (1)

1.0 (l) 1.7 (2)

1.1 (1)

1.0 (1) 1.3 (1)

1 0 (1) 1.1 (1)

1.0 1.0 1.1 1.4 1.9

1.1 (1) 1.9 (2)

(1) (1) (1) (2) (2)

T-15

1.9 (2) 1.1 (l)

1.0 (1)

0.9 (1)

1.1 (1) 1.0 (1) 1.0 (1)

1.1 1.0 1.3 1.1

(1) (1) (1) (,)

1.2 (I) 1.1 (1) 1.0 (1)

0.9 (1)

0.8 (1) 0.6 (1)

3.6 (4)

1.0 (1) 1.2 (I)

1.0 (1)

0.9 (1) 0.9 (1)

1.0 (1)

9 12.9

8 6.9

5 12.1

0.7 (1)

2.0 (2) 1.0 (l)

1.0 (1) 0.9 (1)

3

11 13.8

18.2

1.0 (1) 15 3.0

1.0 (1) 7 18.2

6 8.6

1.0 (1) 6 13.4

1.0 (1) 7 15.2

Results are expa~essed as mol.ar raHos. Values in parentheses are the nearest integers.

T h e p ep¢ide m a p s o f the ~tryptic digests of cMf a n d rat H2B p r e s e n t e d i n figure 1 a p p e a r q u i t e s i m i l a r . Tlais s.imilarity s t r o n g l y snggesCs a s¢ruct u r M i den~tity of t h e I w o 9 r o t e i n s , wh~i,ch w a s e v i d e n c e d b y stru,ctura,1 stt~dies of t h e tryp~ic pepfides.

BIOCHIMIE, 1979, 61, n ° 1.

w e r e d i r e c t l y o b t a i n e d i n p u r e f o r m . T h r e e of t h e m h a d to be p u r i f i e d b y p a p e r c h r o m a t o g r a p h y [11]. T h e a m i n o aci, d c o m p o s i , t i o n a n d y i e l d of t h e t r y p t i e ~eptid,es a r e g i v e n i n table II. B e c a u s e of

S e q u e n c e of rat h i s t o n e H 2 B a n d p h o s p h o r y l a t i o n sites.

67

The i n o r g a n i c [32p] p h o s p h a t e w a s eluted in f r a c t i o n 1. The labelled .peptides, obtMned direcfly i n p u r e form, w e r e a n a l y s e d and l e d us to i d e n t i f y four sites of p h o s p h o r y l a t i o n in the histone H2B (table III).

i n c o m p l e t e cleavage b y t r y p s i n , fo.ur p e p t i d e s (T-3, T-5, T-6 a n d T-10) are accomlaani~ed b y one d e r i v a t i v e (T-3a, T-5a, T-6a a n d T-10,a). The c o m p l e t e amin~) a c i d sequence of the r a t t h y m u s histone H2B (figure 3) w a s d e d u c e d f r o m the s t r u c t u r a l studies of the t r y p t i c p e p t i d e s w h i c h w e r e o r d e r e d b y c o m p a r i s o n w i t h the a m i n o a c i d sequence of the calf hom,ologous histone.

P a r t i a l cleavage b y t r y p s i n was o b s e r v e d at the P

Both histones have an i d e n t i c a l p r i m a r y structure.

A r g - - S e r bond, pr(rducing p e p t i d e T-9a t o g e t h e r 31 32 w i t h p e p t i d e T-8a. This p r o b a b l y due to the acidic e n v i r o n m e n t i n t r o d u c e d b y the p h a s p h a t e groups.

The r a d i o a u t o g r a p h y of the p e p t i d e ,map of the t r y p t i c h y d r o l y s a t e of the p h o s p h o r y l a t e d historic H2B ~labelled w i t h [~eP] p h o s p h a t e s h o w e d five ra,di~oactive spots, once c o r r e s p o n d i n g to i,norganic p h o s p h a t e in excess, t h r e e to n e u t r a l p e p l i d e s a n d one to a bas,ic p e p t i d e u p o n electrophoTesis at

The amo.unts of p h o s p h o r y l a t i o n at Ser-32 (66 p e r cent) a n d Ser-36 (26 p e r cent) r e p r e s e n t t o g e t h e r 92 p e r cent of ;the total amo.u.nt of phosp h a t e incorporat~ed into the r a t h i s t o n e H2B. On the o t h e r h a n d , Ser-87 and Ser-91 ,are m i n o r sites of phosph~)rylafion a n d are e q u a l l y labelled.

A 570 nm NH 3

free Lys

A

[]

radioactive

fractions

Tga 0.5

i

rio T 16

2

T16

34

5

o

6

, 275

B 1.O

free

|

LyS

2 I 275

T8a

7

L 550

I NH3

1 O

/

8 , 825

//

, 1925

//'

I 1925

2,

2 O0 Effluent ( m l )

T16

3 ' 550

4 I 825

5 ?0 2 O Effluent (ml)

FIG. 4. - - Elulion diagram of lryplic peptides from hislone H2B from rat thymus, fraclionaled on Chromobeads P column. A : phosphoryl~ated lit'stone I~B. B native historic H~2~B. " Peptides were des:ign:at.ed by the symbol T and numbered as indicated in t:he legend of figure 1.

p H 3.6. In ion ex,change c h r o m a t o g r a p h y , these l a b e l l e d p e p f i d e s h a d ,an elu4ion volume s m a l l e r to that of t h e i r c o r r e s p o n d i n g u n l a b e l l e d p e p t i d e s as s h o w n in figu.re 4. BIOCHIMIE, 1979, 61, n ° 1.

Ser-32 and Ser-36 are also m a j o r sites of phosp h o r y l a l i o n in calf h i s t o n e H2B w h e n c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e from s i l k w o r m p u p a e [5] or from b o v i n e c e r e b e l l u m [61 is used.

A. Martinage and coil.

68

Ser-36 is preferen,tial~y p h o s p h o r y l a t e d w i t h protein kin~ase f r o m h u m a n t o n s i l i a r 4ymphocytes [17] or f r o m p i g b r a i ~ [18].

As p o i n t e d out by Mac Leod et al. [19] and Small et al. [20], Ser-32 and Ser-36 are l o c a t e d in p r e d i c t e d ~-turn structure. The l o c a t i o n of a phos-

TABLE III. L o c a t i o n o f p h o s p h o s e r i n e r e s i d u e s in rat t h y m u s h i s t o n e H 2 B p h o s p h o r y l a t e d in v i t r o w i t h c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e f r o m rat p a n c r e a s .

Peptide

Sequence of tryptic pepfide

Sites of phosphorylation

P T-8a

I

Lys-Arg-Ser-Arg 32

Ser 32

P

I

T-9a

Ser-Arg 32 P

I

T-10

T-16

Glu-Ser-Tyr-Ser-Val-Tyr-Val-Tyr-Lys 36 P P I [ Ser-Thr- lle-Thr-Ser-Arg 87 91

Thus, the cycli.c AMP.depen.dent p r o t e i n ldnases o b t a i n e d f r o m v a r i o u s s.o~rces do not a p p e a r to be species- or tiss.ue-specific. H o w e v e r , t h e r e is some degree of s p e c i f i c i t y for rat pan,creas p r o t e i n kinas:e w h i c h p h o s p h o r y l a t e s Ser-87 and Ser-91 an~d for p i g b r a i n protein kinase w h i c h phosphorylat,es Ser-14 [18]. T h e p h o s p h o r y l a t e d resid,ues of s.erine at positio,ns 14, 32, 36 and 87 ,are l o c a t e d in a s e q u e n c e B-X-Ser w h e r e B is lysine or .arginine anal X, a bast,c, n e u t r a l or a c i d i c r e s i d u e [18, 19]. T h e r e f o r e the p h o s p h o r y l a f i o n of the serine 91 .appears to be quite atypi,cal and we .cannoi disc.ard the possibility of a small .contamination of o u r p r e p a r a tion of cy.elic AMP-depen,d,cmt p,ro,tein kin~ase by a diff,e r e n t kinase. If w.e c o n s i d e r n o w the p osit~on of the m a j o r sites of phosphoryl, atio.n in the a m i n o acid s e q u e n c e of h i s t o n e H2iB, w e can observe that they are ,located in the a m i n o t e r m i n a l DNA-binding region of the protein, .close to a basic cluster and therefore, the p h o s p h o r y l a t i o n caul.d m o d u late the e l e c t r o s t a t i c i n t e r a c t i o n s of the DNA w i t h the histone. BIOCHIMIE, 1979, 61, n ° 1.

Ser 36

Ser 87 Ser 91

p h o r y l a t e d s e r i n e in a ~-turn is .a.lso f o u n d in calf h i s i o n e H2A (Ser-19) and in t r o u t his.tone H1 (Ser-145, -1.61, -182 [19]. A c c o r d i n g to Mac Leod e t al. [10] the p r o t e i n ki.nase vcou,ld r e c o g n i z e preferential'ly the s.erine residues thus e x p o s e d by this p a r t i c u l a r c.onform.alion.

Aeknowlegments. This w o r k was supported by grant n ° 75.5.059.2 [rom the I n s t i t u t National de la Sant~ et de la Recherche Mddicale and by the Centre National de la Recherche S c i e n t i f i q u e (L.A. n ° 268). W e acknowledge w i t h pleasure the s k i l l f u l technical assistance of Miss A. Hdmez and of Mrs. D. Bela'iche and M. J. Dupire.

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S e q u e n c e o f r a t h i s t o n e H 2 B a n d p h o s p h o r y l a t i o n sites. 6. Kuroda, Y., tIashimoto, E. & N,ishizuka, Y. ; Hamana, K. & Iwai, K. (1976) Bioehem. Biophys. Res. Commun., 71, 629-635. 7. John,s, E. W. (1964) Biochem. J., 92, 55r59. 8. M,artinage, A., Sauti6re, P., Kerckaert, J. P. & Biserte, G. (1976) Biochim. Biophys. Aeta, 420, 37-41. 9. Gros, C. & Laboues, s,e, B. (1'9,69) Eur. J. Biochem., 7, 463-470. 10. Sauti6re, P., Breynaert, M. D., Mosehetto, Y. & B,is.erte, G. (197@,) C. R. Acad. Sci. Paris, D 271, 364-365. 11. Sauti6re, P., M,oschetto, Y., Dauirevaux, M. & Biserte, G. (1970) Eur. J. Bioehem., 12, 222-226. 12. Cohen-Sola], M. a Bernard, J. L. (19'73) J. Chromatog., 80, 140-143. 13. Yamada, S. ~ I,tano, ~ . A. (19.66) Biochim. Biophys. Acta, 130, 538-54~).

BtOCHIMIE, 1979, 61, n ° 1.

69

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