Phosphorylation of protein B-50 (GAP-43) from adult rat brain cortex by casein kinase II

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

Vol. 155, No. 3, 1988

Pages 1207-1212

September 30, 1988

PHOSPHORYLATION OF PROTEIN B-50 (GAP-43) FROM R A T B R A I N C O R T E X B Y C A S E I N K I N A S E II

ADULT

Michael R. Pisano, Mohammed G. Hegazy, Erwin M. Reimann and Linda A. Dokas* Departments of Biochemistry and Neurology* Medical College of Ohio, Toledo, OH. 43699

Received July 18, 1988

Summary: The phosphoprotein B-50 (GAP-43) was purified from adult rat brain cortex and phosphorylated by casein kinase II. Phosphorylation of B-50 by casein kinase II approached 1.2 mol phosphate/mol B-50. The apparent K m of casein kinase II for B-50 was 4 ~M with an apparent Vma x of 13 nmol.min-l.mg -I. A tryptic p h o s p h o p e p t i d ~ 2 m a p on r e v e r s e d phase HPLC and phosphoamino acid analysis of [ P]B-50 showed that casein kinase II p h o s p h o r y l a t e d zn serlne residue(s) which were located in a single tryptic peptide. Phosphorylation of B-50 by casein kinase II was inhibited more than 90% by 5 ~g heparin/ml or 2.4 mM peptide substrate specific for casein kinase II (RRREEETEEE). The initial p h o s p h o r y l a t i o n rate was increased about 2-fold by 1 mM spermine. © 1988 Academic Press, Inc.

B-50

(also known

phosphoprotein system function been

(3) of

(1,2).

and

to

The

appears

synaptic

proposed

as GAP-43)

to

protein be

relations

occur

at

is a presynaptic is

specific

involved (4).

multiple

in

the

membrane-bound to

(5).

nervous

development

Phosphorylation sites

the

of B-50

However,

and has

among

a

*This study was supported by NIH grants NS 23598, HL 36573 and DK 19231. We thank C h r i s Yunker for his assistance in the purification of CK-II and Don Rifner for help with the photography. Drs. Henk Zwiers and Willem H. Gispen are acknowledged for their help with the method for purification of B-50. Abbreviations: CK-II, casein kinase-II; GAP-43, growth associated protein (apparent M r = 43,000); SDS, sodium dodecyl sulfate; TPCK, L-l-(tosylamido)-2 phenylethyl chloromethyl ketone; TCA, trichloroacetic acid; CK-II peptide, substrate specific for casein, kinase-II (RRREEETEEE); HPLC, high performance liquid chromatography.

1207

0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 155, No. 3, 1988

number

of

activated, C),

was

protein

(6).

shown

to

suggesting

kinases

examined

phospholipid-dependent

able

brain

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to

significantly

Phosphorylation inhibit a role

i_nn vitro,

protein

kinase

phosphorylate

of B-50

in

from

kinase

in the

synaptic

Ca +2kinase

adult C has

4-phosphate

for B-50 phosphorylation metabolism

the

(protein

B-50

by protein

phosphatidylinositol

polyphosphoinositide

only

rat been

kinase,

regulation

plasma

of

membranes

(4,7). Casein independent

kinase protein

acidic proteins

sites

amino

for

(CK-II)

kinase

(8,9).

including brain contains

II

Since B-50 is an acidic protein

(12) ana

we

Phosphorylation were located

presented

B-50

to

of

B-50

phosphorylates tissues,

(13,14),

results

nucleotide-

in most mammalian

B-50 may serve as a substrate The

and cyclic

preferentially

is found

acid sequences

CK-II

phosphorylated

that

CK-II

(i0,ii).

is a Ca +2

the by

that are potential investigated

the

phosphorylation possibility

that

for CK-II. in

this

study

extent

of

1.2

CK-II

was

in

indicate mol

of

serine

that

CK-II

32p/mol

B-50.

residues

which

in a single tryptic peptide.

MATERIALS

AND

METHODS

Phosphorylation of B-50: Unless otherwise indicated, B-50 (0.6 ~M) was incubated at 30 ° in a 50 ~i assay mixture containing 20 mM Tris-HCl (pH 7.6), 0.2 mM EDTA, 40 ~M EGTA, 1 mM KF, 20 mM 2mercaptoethanol, 4 ~M cAMP-dependent protein kinase inhibitor peptide and the appropriate units of CK-II. The reaction was started by the addition of a mixture containing m a g n e s i u m acetate and ATP to give a final concentration of i0 mM m a g n e s i u m and 0.2 mM ATP (500-1000 cpm/pmol). The reaction was stopped by spotting 20 ~i aliquots onto phosphocellulose papers (P81) and the papers were processed (15). Radioactivity incorporated into B-50 was determined by liquid scintillation counting. Data presented were corrected for autophosphorylation of CK-II. Where indicated the reactions were stopped by the addition of sample preparation buffer that contained 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, 0.1% bromphenol blue and 62.5 mM Tris-HCl, pH 6.8 and were applied to SDS-polyacrylamide gels following the method of Laemmli (16). The B-50 bands were excised, placed in scintillation fluid and counted for radioactivity. Tryptic Phosphopeptide Map and Phosphoamino Acid Analysis: The standard assay for phosphorylation of B-50 by CK-II was carried out and a 5 ~i aliquot of the reaction mixture was spotted onto • JZ phosphocellulose paper to determlne P incorporated into B-50 as described above. The rest of the reaction mixture was diluted with an equal volume of ice cold 20% TCA and the sample was placed on ice for 1 hour. The precipitate was collected by centrifugation, the pellet was washed with ice cold 5% TCA, then with cold ether and dried. The pellet was dissolved in 50 ~i of 8.0 M urea and then diluted to 2.0 M urea with 0.i M ammonium bicarbonate, pH 8.2. TPCK-trypsin (Worthington) was added to a 1208

Vol. 155, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

final c o n c e n t r a t i o n of 1 mg/ml and the sample was incubated at 30 ° for 7 hours. Thirty ~i of 70% formic acid was added and the sample was applied to a C18 reversed phase HPLC column (Linden, Indiana). A linear gradien£ from 0.1% t r i f l u o r o a c e t i c acid in H20 to 0.09% t r i f l u o r o a c e t i c acid in 60% a c e t o n i t r i l e was applied at a flow rate of 1 ml/min. Fractions of 1.0 ml were c o l l e c t e d and counted for r a d i o a c t i v i t y by Cerenkov counting. The peak fraction (fig. 4) was dried and subjected to p h o s p h o a m i n o acid analysis as d e s c r i b e d (17). Other Methods: B-50 was isolated from adult rat brain cortex by a method of alkaline extraction and ammonium sulfate p r e c i p i t a t i o n (5). CK-II was purified from rabbit skeletal muscle as p r e v i o u s l y d e s c r i b e d (18). One unit of CK-II a c t i v i t y was defined as the amount of the enzyme that catalyzes the i n c o r p o r a t i o n of 1 nmol of 32p~ into CK-II p e p t i d e per min Protein c o n c e n t r a t i o n was d e t e r m i n e d by the m e t h o d of H a r t r e e (19) or Bradford (20) using bovine serum albumin as standard. B-50 c o n c e n t r a t i o n was d e t e r m i n e d by d e n s i t o m e t r i c scanning of fast green stained gels using known amounts of bovine serum albumin on the same gels as standards (5). Scanning was p e r f o r m e d on a LKB ZEINEH soft laser scanning densitometer. B-50 has been shown to m i g r a t e a n o m a l o u s l y (apparent M r = 48,000) on SDSp o l y a c r y l a m i d e gels (21). The molar c o n c e n t r a t i o n of B-50 was determined using M r 24,000 for the protein based on the amino acid c o m p o s i t i o n as deduced from the nucleotide sequence of B-50 cDNA (13). A u t o r a d i o g r a p h y was p e r f o r m e d as d e s c r i b e d (18). The values of apparent K m and Vma x were d e t e r m i n e d as in (22). The inhibitor p e p t i d e o f c A M P - d e p e n d e n t p r o t e i n kinase (23) was a generous gift of Dr. Edwin Krebs, Seattle, Washington. [~-32p]ATP was p u r c h a s e d from ICN and spermine t e t r a h y d r o c h l o r i d e from Sigma. CK-II peptide (RRREEETEEE) was o b t a i n e d from A p p l i e d Biosystems and p r o t e i n standards were p u r c h a s e d from BioRad.

RESULTS

AND

DISCUSSION

When B-50 was incubated with CK-II at 30 ° in the p r e s e n c e of [~-32p]ATP,

the

32p/mol

within

B-50

time resulted 1.2

mol

B-50

n m o l . m i n - l . m g -I .

r e l a t i v e l y good peptide

was

120

These

min.

0.6

mol

the

incubation

of B-50 that

approached

In

the

absence

into

B-50

4 ~M with

values

nearly

Increasing

incorporated

for B-50 was

substrate

CK-II

of

(fig.

CK-II, i).

The

an apparent Vma x of

indicate

that

B-50

is

a

for CK-II compared to casein and CK-II

activity

c o n c e n t r a t i o n s of heparin tested the effects by

i).

(24). Since

B-50

incorporated

(fig.

within

no p h o s p h a t e

apparent K m of CK-II 13

kinase

i0 min

in further p h o s p h o r y l a t i o n

32p/mol

virtually

protein

CK-II.

is

and spermine

Phosphorylation

not shown).

rate was

to

be

inhibited

(25) and increased by spermine

of heparin

of

more than 95% by 5 ~g heparin/ml phosphorylation

known

assay

increased

Phosphorylation

B-50

of B-50

1209

by

low

(26), we

on p h o s p h o r y l a t i o n

by (fig.

CK-II

was

inhibited

i), but the

initial

2-fold by 1 mM spermine by CK-II

was

also

of

(data

inhibited

Vol. 155, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

i.4

1.61 A

i.2

f

¢-n

O.a 0.5 0.4 0.2

(D 0.0 o

E

~

0.8

~

0.4

0 . 0

20

60 80 TIME (MIN)

40

100

'

120 ( ~ ~

Fig. i. Time course of B-50 #M) was incubated with CK-II or presence (A) of 5 ~g Phosphorylation of B-50 in conditions were as described

"

'

0.0

'

"

"

'

'

"

"

"

'

"

"

"

'

"

"

"

'

0.4

0.8 1.2 1.6 2.0 CK-II PEPTIDE (mM)

phosphorylation (0.17 units/ml) heparin/ml of the absence of in Materials and

by CK-II. B-50 (0.6 in the absence (m) assay mixture. (o) added CK-II. Assay Methods.

"

"

"

'

2.4

Fig. 2. Effect of CK-II peptide on phosphorylation of B-50 by CKII. B-50 (0.6 #M) was incubated with CK-II (0.09 units/ml) in the presence of the indicated concentrations of CK-II peptide. The standard assay conditions were employed. After a 30 min incubation, the reactions were stopped by the addition of sample preparation buffer and the proteins were separated on 11% SDSpolyacrylamide gels. The B-50 bands were excised and counted for radioactivity as described in Materials and Methods.

up to 94% b y (fig. by

2).

including

These

CK-II

Figure of

B-50

results

and

was

contaminating

CK-II

the

the CK-II

resulted

in

highly

incorporated A tryptic

50

(fig.

3B).

CK-II

revealed

that

a

single

peptide

that

acetonitrile

(fig.

II p h o s p h o r y l a t e d It acidic that third

most

Examination the

serine

192 are

these

positions

two n+3

eluted

the

n+4,

that to

to

protein

kinase

of B - 5 0

phase

residues

acidic

structure that

residue

145

has 192

B-

21%

that

CK-

3C).

cluster

residues

residue of

B-50

serine

in

at

(fig. a

residue

only

B-50

HPLC

showed

recognizes

phosphorylated

with

the

phosphorylated

analysis

is an

serine

that

associated

phosphorylated

b y an a c i d i c

1210

and

phosphorylated

in s e r i n e CK-II

primary

serine

preparation

acid

indicates

whereas

for t h e p u r i f i c a t i o n

reversed

the

the

the (13)

used

map

determinant

residues, and

from

exclusively

sequence followed

phosphorylated

another

kinase

Phosphoamino

documented

of

was

was mostly

phosphopeptide

C-terminal

from

cDNA

by CK-II

C-terminal

position

B-50

of

purified

the protein

important

27).

Of

B-50

is w e l l

residues

the

4).

result

the p r o c e d u r e

radioactivity

by

that

assay

preparation.

that a

in t h e p h o s p h o r y l a t i o n

demonstrate

not

3A shows

peptide

in

of and the

(14,24deduced 145

and

in t h e n+3 p o s i t i o n . acidic

has

acidic

residues residues

at at

Vol. 155, No. 3, 1 9 8 8

2. 3. 4. 5.

6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Sorenson, R.G., Kleine, L.P. and Mahler H.R. (1981) Brain Res. Bull. 7, 57-61. Kristjansson, G.I., Zwiers, H., Oestreicher, A.B. and Gispen, W.H. (1982) J. Neurochem. 39, 371-378. Benowitz, L.I. and Routtenberg, A. (1987) TINS i0, 527532. Zwiers, H. Verhaagen, J., van Dongen, C.J., De Graan, P.N.E. and Gispen, W.H. (1985) J. Neurochem. 44, 10831090. Aloyo, J.V., Zwiers, H. and Gispen, W.H. (1983) J. Neurochem. 41, 649-653. Van Dongen, C.J., Zwiers, H., De Graan, P.N.E. and Gispen, W.H. (1985) Biochem. Biophys. Res. Commun. 128, 1219-1227. Hathaway, G.M. and Traugh, J.A. (1982) Curr. Top. Cell Regul. 21, 101-127. Pinna, L.A., Meggio, F., Marchiori, F. and Borin, G. (1984) FEBS Lett. 171, 211-214. Singh, T.J. and Huang, K.P. (1985) FEBS Lett. 190, 8488. Nairn, A.C., Hemmings, H.C. Jr. and Greengard, P. (1985) Ann. Rev. Biochem. 54, 931-976. Zwiers, H., Schotman, P. and Gispen, W.H. (1980) J. Neurochem. 34, 1689-1700. Karns, L.R., Ng, S.C., Freeman, J.A. and Fishman, M.C. (1987) Science 236, 597-600. Marin, O., Meggio, F., Marchiori, F. Borin, G. and Pinna, L.A. (1986) Eur. J. Biochem. 160, 239-244. Roskoski, R. (1983) Methods Enzymol. 99, 3-6. Laemmli, U.K. (1970) Nature (London) 227, 680-685. Hegazy, M.G., Thysseril, T.J., Schlender, K.K., and Reimann, E.M. (1987) Arch. Biochem. Biophys. 258, 470481. Hegazy, M.G., Schlender, K.K. and Reimann, E.M. (1987) Biochim. Biophys. Acta 927, 269-279. Hartree, E.F. (1972) Anal. Biochem. 48, 422-427. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. Benowitz, L.I., Perrone-Bizzozero, N.I. and Finkelstein, S.P. (1987) J. Neurochem. 48, 1640-1647. Cleland, W.W. (1967) Adv. Enzymol. 29, 1-32. Scott, J.D., Glaccum, M.B., Fischer, E.H. and Krebs, E.G. (1986) Proc. Natl. Acad. Sci. (USA) 83, 1613-1616. Kuenzel, E.A., Mulligan, J.A., Sommercorn, J. and Krebs, E.G. (1987) J. Biol. Chem. 262, 9136-9140. Meggio, F., Pinna, L.A., Marchiori, F. and Borin, G. (1983) FEBS Lett. 162, 235-238. Hathaway, G.M. and Traugh, J.A. (1984) J. Biol. Chem. 259, 7011-7015. Meggio, F., Marchiori, F., Borin, G., Chessa, G. and Pinna, L.A. (1984) J. Biol. Chem. 259, 14576-14579. DePaoli-Roach, A.A., (1984) J. Biol. Chem. 259, 1214412152. Meggio, F., Flamigni, F., Caldarera, C.M., Gualtieri, C. and Pinna, L.A. (1984) Biochem. Biophys. Res. Commun. 122, 997-1004. Woodget, J.R. and Cohen, P. (1984) Biochim. Biophys. Acta 788, 339-347. Ackerman, P., Glover, C.V. and Osheroff, N. (1985) Proc. Natl. Acad. Sci. (USA) 82, 3164-3168. Corvera, S., Roach, P.J., Depaoli-Roach, A.A. and Czech, M.P. (1988) J. Biol. Chem. 263, 3116-3122.

1212

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

C +

A

~.-p-Ser B

40

3O

• 4B-50

~

o

-K,-p-T h r

x

20

~0

ORIGIN

0

~0

(i)

20 30 40 50 RETENTION TIME (MIN)

60

Fig. 3. A) Fast green stained 11% SDS-polyacrylamide gel. Lane i, protein standards from top to bottom: phosphorylase b (97.4 kDa), bovine serum albumin (68 kDa), ovalbumin (43 kDa), carbonic anhydrase (31 kDa) and soybean trypsin inhibitor (21 kDa); lane 2, preparation of purified B-50 (0.4 ~g). B) Autoradiogram of B50 phosphorylated by CK-II for 30 min as shown in figure i. C) Phosphoamino acid analysis of B-50 phosphorylated by CK-II. Phosphorylation of B-50 by CK-II and phosphoamino acid analysis were performed as described in Materials and Methods. Positions of phosphoserine (p-Ser) and phosphothreonine (p-Thr) standards are indicated. Fig. 4. Tryptic phosphopeptide map of B-50 phosphcrylated by CKII. B - ~ (3.04 ~M) was3~hosphorylated by CK-II (0.09 units/ml) to 1 mol ~ZP/mol B-50. [ P]B-50 was subjected to complete tryptic digestion and the tryptic peptides were separated on reversed phase HPLC as described in Materials and Methods.

positions serine

n-2,

n+3,

n+4

and

n+5.

192 is the p r i m a r y t a r g e t

Therefore,

site in B-50

we

suggest

that

for p h o s p h o r y l a t i o n

by CK-II. B-50 (6). to

is k n o w n to u n d e r g o p h o s p h o r y l a t i o n

Phosphorylation inhibit

not

CK-II

yet is

significance been

widely

phosphorylates phosphorylation function

by

protein

phosphatidylinositol

physiological has

of B-50

of

determined.

a

number

(28-32),

of we

4-phosphate

phosphorylation However,

distributed

by protein kinase

kinase

in

taking

brain

proteins suggest

that

(Ii) that CK-II

C has

been

kinase

(7).

of

by

B-50

into and are may

that CK-II by

modulate

the

of t h i s protein.

Zwiers, H., O e s t r e i c h e r , A.B., Bisby, M.A., De Graan, P.N.E. and Gispen, W.H. (1987) A x o n a l T r a n s p o r t (M.A. B i s b y and R.S. Smith, Eds.), pp. 385-406. Liss, N e w York. 1211

CK-II

regulated

REFERENCES

I.

The

account that

C

shown