Phosphorylation of myelin basic protein and peptides by ganglioside-stimulated protein kinase

Vol.

165,

No.

November

BIOCHEMICAL

1, 1989

30,

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

1989

93-100

PHOSPHORYLATION OF MYELIN BASIC PROTEIN AND PEPTIDES BY GANGLIOSIDE-STIMUL

PROTEIN KINASE

Kai-Foon Laboratory Received

of Experimental September

18,

Jesse

Chan NINDS,

Neuropathology,

NIH,

Bethesda,

MD 20892

1989

by a Summary: Rabbit myelin basic protein (MBP) was phosphorylated ganglioside-stimulated protein kinase to a stoichiometry of 1.4 and 2.1 mol phosphate/m01 MBP in the presence and absence of G,,,, respectively. Tworevealed that two of the sites of dimensional peptide mapping analyses phosphorylation were distinct from those catalyzed by CAMP-dependent protein kinase or protein kinase C. Phosphorylation of one of these sites by ganglioside-stimulated protein kinase was inhibited by G,,,, suggesting that the inhibitory effect of gangliosides on MBP phosphorylation may be substrate-directed. Although ganglioside-stimulated protein kinase did not phosphorylate MBP at a domain containing residues 82-117, a synthetic peptide Arg-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys corresponding to residues 111-120 was phosphorylated by the kinase in a ganglioside-stimulated manner. These findings suggest that the conformation of MBP may be important in determining its phosphorylatability.
Myelin total

basic

protein

in

cytoplasmic

side

molecule

the

catalyzed

conduction

(g-10).

major

kinase

(11-15,

several

seryl

dense

and

include

been

and threonyl

event of

protein Although residues

may act

MBP under

these

C,

(14),

covalent

--in vitro protein

myelin's

modification vivo --In of axons

depolarization

this

modification

conditions

can

be

phosphotransferases

a Ca"'/phospholipid-dependent

kinase, two

of

mechanisms. Both

the

as an adhesion

post-translational (8).

kinase

on the

located

maintenance

to enhance

protein

is

and

dephosphorylation shown

of

It

and

undergo

one-third

(1,2).

(3-7)

and Ca2'- independent

and CAMP-dependent 17-18).

myelin

line

a dynamic

have

approximately

compaction,

Phosphorylation

These kinase,

system

MBP can

by CaX' -dependent

(11-19). protein

the

formation,

of MBP is

impulse

comprises

nervous

phosphorylation

phosphorylation process

(MBP)

structure.

through and

central of

for

multilamellar

protein

a Ca2'-independent

kinases they

still

can phosphorylate cannot

account

protein MBP at for

all

Abbreviations: MBP, myelin basic protein; HEPES, N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid; EGTA, ethyleneglycol bis(R-aminoethyl ether)N,N,N',N'-tetraacetic acid; CAMP, adenosine-3':5'-monophosphate. Designation of ganglio-series gangliosides is based on the Svennerholm system (23).

93

0006-291X/89 $1.50 Copyright 0 I989 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 165, No. 1, 1989 the

endogenous

BIOCHEMICAL

phosphorylation

Recent

investigations

profound

effects

on

the

proteins

(17).

A

novel

identified

and partially

whether

this

also

play

phosphorylate in

a

presence from

protein

kinase. may

These

of

from

in

that

of

several

protein pig

brain

myelin this

it

kinase (21).

by

findings

Two of protein

shown

that

modification

for

NATERIALS

AND

the

protein

C and

this

kinase,

ability

phosphorylation

kinase

suggest

in the

the

been

To investigate protein

by ganglioside-stimulated G,,,.

myelin has

its

is

have

other

function,

report,

MBP (20).

gangliosides

a Ca"' -independent

modulating In

rabbit

MBP and

guinea

enzyme,

catalyzed

be important

purified

ganglioside-stimulated purified

absence

those

in revealed

phosphorylation

role

or

found

laboratory

MBP can be phosphorylated

distinct reaction

this

MBP was studied.

that the

sites

ganglioside-dependent

may

time

in

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

first kinase

sites

by

protein

to

are

CAMP-dependent phosphorylation

of MBP.

MlXTHODS

Rabbit myelin basic protein was obtained from Calbiochem. The purity of the protein was monitored by using high-performance liquid chromatography (HPLC) and high-performance capillary electrophoresis techniques. A ganglioside-stimulated protein kinase was partially purified from guinea pig brains as previously described (21). A membrane-associated protein kinase C and the catalytic subunit of CAMP-dependent protein kinase were purified to apparent homogeneity according to previously reported procedures with minor modifications (18, 22). G,,,, was obtained from Bachem, Inc. Synthetic peptides including an encephomyelitogenic peptide Phe-Ser-Trp-Gly-Ala-Gluand Leu-Arg-Arg-Ala-Ser-Leu-Gly Gly-Gln-Arg, MBP residues 68-84 (guinea pig), were purchased from Peninsula. A peptide with the sequence of Arg-Phe-SerTrp-Gly-Ala-Glu-Gly-Gln-Lys custom synthesized and purified to was homogeneity by using high-performance liquid chromatography. Phosphorylation of MBP (0.4 mg/ml) or synthetic peptides by different protein kinases was carried out at 30 OC in reaction mixtures containing 50 mM HEPES, pH 7.4, 10 mM magnesium acetate, and 167 uM [7-S"P]ATP. The reaction catalyzed by the partially purified ganglioside-stimulated protein catalyzed by kinase contained 266 ug/ml G,,,, and 1.0 mM EGTA; the reaction the catalytic subunit of CAMP-dependent protein kinase (2.3 pg/ml) contained 1.0 mM EGTA; and the reaction catalyzed by protein kinase C contained 0.4 mM calcium chloride and 40 pg/ml phosphatidylserine. At timed intervals, aliquots were taken and 32P incorporation into MBP was monitored by using In other experiments, the phosphocellulose P-81 paper assays (17, 21). reactions were terminated with 20% (w/v) ice-cold trichloroacetic acid (TCA). The precipitated MBP was centrifuged and washed with 5% TCA (3 times) and The sites of phosphorylation in MBP were anhydrous ethyl ether (3 times). analyzed by using two-dimensional peptide mapping techniques after digestion reversed-phase HPLC after digestion with with trypsin (17, 21) or by using Staphylococcus aureus V8 protease as described previously (18).

RESULTS

A ganglioside-stimulated pig

brain

substrates This

protein

could such

undergo

AND

DISCUSSION

protein

kinase

autophosphorylation

as Leu-Arg-Arg-Ala-Ser-Leu-Gly

kinase

also

phosphorylated

partially in the

MBP isolated 94

purified

and phosphorylation presence from

rabbit

from

guinea

of exogenous of

G,,,

(21).

brain

(Fig.

Vol.

165,

No.

1, 1989

BIOCHEMICAL

3

20

AND

I

I

40

60

BIOPHYSICAL

RESEARCH

1

I

80

TIME

COMMUNICATIONS

1

100

120

(MIN)

Phosphorylation of rabbit MBP by ganqlioside-stimulated protein Fig. 1. kinase Rabbit MBP (0.4 mg/ml) was phosphorylated by a partially purified aoside-stimulated protein kinase in the presence (*) or absence (0) of G.,.lh (266 pg/ml) under conditions similar to those described under Materials At timed intervals, and Methods. "=P incorporation into MBP was monitored by using phosphocellulose P81 paper assays. The experiments were repeated three times and the results of one representative experiment are shown.

1).

However,

The

stoichiometry

1.4

and

phosphorylation

2.1

""P/m01

mol

seemingly

opposite

protein

had MBP. the

kinase no

of also

MBP

Approximately

4.3

and absence

I.

Protein

on

even

presence

respectively

Effects rabbit

by

when and

(Table by

the

the

was inhibited effects

Table

the

attenuated

effects

inhibitory

presence

observed

absence

I),

G,,,,.

was

absent.

of

indicating

Thus,

enyzmic

of my&in

catalytic

by G,,,

on protein and 4.0

G,,,,

was

that

the

gangliosides

activity

of GT,k,,

gangliosides basic

subunit

(Table

kinase

respectively

I).

of

can

ganglioside-

of

were

incorporated

(Table

(mol

gangliosides

phosphorylation into

of MBP in

I).

on the stoichiometry of protein by various protein

Stoichiometry

CAMP-dependent

However,

C-catalyzed

phosphate

mol

kinases

"P/m01

phosphorylation kinases

of

MBP)

-GT,b PKJ PKA PKC

GT1,

kinase.

Phosphorylation protein

was

was in

MBP,

reaction

stimulated

MBP

of phosphorylation

phosphorylation exert

of

+G, 1L>

2.12

r

1.93

+ 0.37

0.38

4.00

+ 0.51

1.39 1.44 4.34

k f +

0.24 0.50 0.56

Rabbit MBP (0.4 mg/ml) was phosphorylated by different protein kinases with or without the presence of 266 pg/ml G,,,, under conditions similar to those described under Materials and Methods. The stoichiometry of phosphorylation was determined after 100 min of reaction as described in Fig. 1. The protein kinases were: ganglioside-stimulated protein kinase (PKJ); the catalytic subunit of CAMP-dependent protein kinase (PKA); and protein kinase C (PKC). 95

Vol. 165, No. 1, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PKJ

PKA

A

PKC

B

C

F

ELECTROPHORESIS

Fig. 2. Two-dimensional peptide mapping of "zP-labeled rabbit MBP phosphorylated by qanglioside-stimulated protein kinase, CAMP-dependent protein kinase or protein kinase C. Rabbit MBP was phosphorylated by ganglioside-stimulated protein kinase (A and D), the catalytic subunit of CAMP-dependent protein kinase (B and E), or protein kinase C (C and F) in the absence (A to C) or presence (D to F) of G,,, (266 ug/ml) as described in the legend to Table I. The '7P-labeled proteins were digested with trypsin and analyzed by using two-dimensional peptide mapping technique. High-voltage electrophoresis was carried out in a buffer containing pyridine/acetic acid/ water (1:10:189; v/v) and ascending chromatography was carried out in a buffer comprised of l-butanol/acetic acid/water/pyridine (12:15:3:10; v/v). The tracking dye was phenol red. The radioactive phosphopeptides were located by using autoradiography.

Analyses peptide

of the

mapping

sites

techniques

ganglioside-stimulated protein

and

(sites

stimulated

the

reaction site

3 was

indicate

that

substrate-directed, phosphorylation To gain

more

2A).

by

trisected

at glutamic

specificity

no

inhibited

substrate

2).

kinase kinase

C (Fig. effects

by gangliosides

due

of to

2C). on

Addition

site

3

4 were of GTIL)

MBP phosphorylation

(Figs.

of

of site

phosphorylation 2D and

MBP phosphorylation

binding

by ganglioside-

2B) whereas

However,

2F).

tryptic

phosphorylation (Fig.

of

CAMP-dependent

""P-labeled

phosphorylated

apparent

modulation

from Two

In contrast,

C (Fig.

by

gangliosides

at

of both 2E). G,,,, or

These may near

be the

1 and 3 of MBP.

information

catalyzed

the

preferentially

had

perhaps sites

two-dimensional

distinct

C (Fig.

protein

kinase

in MBP by using was

by protein

mixtures

by protein

1 and

results

were (Fig.

phosphorylated

catalyzed site

kinase

by CAMP-dependent

specifically to

kinase

that

kinase

protein

1 and 2)

protein

was preferred

revealed

protein

kinase

peptides

of phosphorylation

on the

gangliaside-stimulated acid

residues

location

of the

protein

kinase,

into

96

three

domains

sites

of phosphorylation

phosphorylated

MBP was

(designated

A, B, and

Vol. 165, No. 1, 1989 C,

BIOCHEMICAL

respectively)

4.0

by digestion

as previously

into

three

3A).

Only

peak

I

and

III

that

peak

may correspond

to

of

fractions

revealed

3B).

MBP,

Other

in the

absence

Thus,

that

18-20)

domain

including

encephalitogenic kinase

highly

conserved

does not

111-120

different

to

peak

identification

of

experimental

allergic was

Gly-Gln-Lys

I

Ser

I

the

II).

Ser-113

residue

may

et

phosphorylated

al.

(14)

dependent

be

protein

in

3A).

domain

B of

MBP.

(Fig.

3B).

sites

(13-15, allergic

MBP at this

corresponding

to

to

isolated

of

the

phosphorylation

al.

aspect.

(13)

have

determining allergic

kinase the

substrate

that

protein

kinase,

not

an

a

These be

substrate by kinetics

Ser-115 the

results

accessible

amino-terminal

in

phosphorylation of

protein

to

was observed

detailed

that

C and this

basic

using

(Table

II).

side

of the

gangliosidestudies

bovine

MBP

reaction protein

are is

may be to

induce

Subsequent studies by Kishimoto et of this site is preferred by CAMP-

phosphorylation rather

for

was stimulated

recognition

more

antigenicity

encephalomyelitis.

revealed

to the

suggested

the

time

mixture.

as

Of course,

this

3C)

Arg-Phe-Ser-Trp-Gly-Ala-Glu-

MBP may

two positions

kinase.

(Fig.

supports

retention

No phosphorylation

for

by

min)

result

The

reaction

rabbit

kinase.

(35.07

by

peptide

Ganglioside-stimulated

peptide rate

G,,,

synthetic

Phe-Ser-Trp-Gly-Ala-Glu-Gly-

shown).

synthetic

phosphorylatability

This

peptide

not

important

by protein in

I was

an

time

(Fig.

of

located

to ascertain

Turner

of peak

of MBP phosphorylation,

this

retention

Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Arg

stimulated

was increased

isolated

and its of

min)

protein

Arg residue

experimental

its

The initial

by addition

the

important

as

phosphorylate

that

required

(34.19

(results

(Table

peptide

Thus,

(Fig.

ganglioside-stimulated

specificity

encephalitogenic

ganglioside-stimulated a

Peaks

C (residues

experimental

112-120),

Analysis

that

min

lo-fold

suggest

an

to phosphorylate

site

tested.

peak

35.02

could

II

phosphorylation

MBP was synthesized

kinases HPLC revealed

similar

to

the

of rabbit

protein

reversed-phase

6-

(residues

B).

gangliosides

within

114)

chromatographic

peak

Arg-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys

residues

kinase

of

unique

radioactivity

of

absence

located

to be able

seem

investigate

oligopeptide

Gln-Arg

residue

This

(domain the

contained

potential

separated

(position

No phosphorylation

or

four

nm.

and domain of

pH

region.

To further

was

B contains

l-81)

III

shown).

presence

determinant

protein

and

280

at

HPLC (Fig.

residue

82-117

phosphorylation

not

the

a seryl

residues Analyses

II

that

(results of

at Trp

A (residues

peaks

indicated

regardless

absorbance

(18).

only

of G,,,

although

domain

were

reversed-phase

of a single

I represents

V8 protease

peptides

by using

highest

respectively

studies

observed,

the

aureus

'ZP-labeled

(I-III)

due to the presence

in MBP and suggests 118-168)

The

peaks

showed

is

Staphylococcus

(18).

polypeptide

characteristics II

with

described

major

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

than

protein 97

kinase

C.

As shown

in

Table

Vol.

165,

No.

1, 1989

BIOCHEMICAL

A2l4

C

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

nm

A2eomn

A214nm

I

,

, 20

I

,

0

30

I 40

1

1

c

50

TIME MN)

Fig. MBP.

3. Localization MBP (5 wg) was

of the sites of phosphorylation in the three domains of phosphorylated by ganglioside-stimulated protein kinase .'"P/mol MBP in the absence of G,,,. The ‘i;;-a stoichiometry of 1.9 mol radioactive MBP was digested by Staphylococcus aureus V8 protease at pH 4.0 and the "P-labeled peptides were analyzed by reversed-phase HPLC on a Vydac Cl8 column using a linear gradient of O-60% (v/v) acetonitrile in 0.05% trifluoroacetic acid (v/v), commencing after 5 min of washing. The elution nm (AUFS=O.S) and at 280 nm (AUFS=O.l) (A). profiles were monitored at 214 Fractions of 0.4 ml were collected and the amounts of radioactivity were The elution profile of a determined by liquid scintillation counting (B). synthetic peptide Arg-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys which corresponds Phosphorylation of this to residues 111-120 in rabbit MBP is shown in C. peptide (1.0 mg/ml) by ganglioside-stimulated protein kinase is summarized in Table II.

98

BIOCHEMICAL

Vol. 165, No. 1, 1989 Table

II.

Comparison

of

substrate various

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

specificities protein kinases

Activity Substrates

of

(cpm

MBP and

peptides

per min)

incorporated PKA

PKJ

for

PKC

+

& Rabbit MBP RFsWGAEGQK LRRASLG

FSWGAEGQR MBP #68-84

10,500

12,400 1,400 4,300

32,300

13,400 25,400 40

(G.

pig)

180,000 60

3,900 9,900

10,400 30 20

30 80

10

20 50

mg/ml) and various synthetic peptides (1.0 protein kinase (PKJ), CAMP-dependent protein kinase (PKA) and protein kinase C (PKC) was carried out at 30 'C for 5 min in reaction mixtures similar to those described under Materials and Methods. The peptide substrates were: RFSWGAEGQK, Arg-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys; LRRASLG, Leu-Arg-Arg-Ala-Ser-Leu-Gly; FSWGAEGQR, Phe-Ser-Trp-Gly-Ala-Glu-GlyHis-Tyr-Gly-Ser-Leu-Pro-Gln-Lys-SerGln-Arg; and MBP #68-84 (guinea pig), Gln-Arg-Ser-Gln-Asp-Glu-Asn-Pro. Phosphorylation

of

rabbit

MBP (0.4

mg/ml) by ganglioside-stimulated

CAMP-dependent protein kinase II, Arg-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys was a poor

substrate

(0.4

mg/ml)

or

(1.0

mgjml)

(Table

et

which

(14) phosphorylated exact

of

newly

biosynthesized

conformation

that

membranes;

and

multilamellae states

or membrane

multiple

sites,

it

on the

conformation

isolated protein assumes residues explain

MBP is

is

not

structure 111-120

(Table

why Thr-98

in

C or

peptide

corresponding

for

possible

3),

may

not

be

MBP

by

the

myelin

to

II).

MBP is

protein

kinase

sequence

of 99

97-103

the

myelin of

the

metabolic

the

by

although

location Ser-113

in

ganglioside-stimulated

peptide not

sites

exact

phosphorylatable

(14,

at

different

and the

conformational

and bovine

a

regions of

Thus,

synthetic

This

specialized

of

multilamellae.

the

acquire

MBP can be phosphorylated

phosphorylated

similar

to

modulation

formation

may become

remains

phosphorylation

different

Because

still

protein into

the

of

kinases

phosphorylation

of

site

to the

to MBP

of Kishimoto

MBP

(i)

the

MBP in

for

that

this

human

peptide

compared

phosphorylation protein

insertion of

stages

CAMP-dependent

bovine

of

facilitate

significantly

(Fig.

kinase

may

of myelin.

various

this

results

two possibilities:

a mechanism

of MBP within

kinase a

at least

necessary

stability

when the

in

or other

phosphorylation

may provide

may depend or

is

support

Ser-115

kinase

MBP

(ii)

In contrast,

peptide

of Leu-Arg-Arg-Ala-Ser-Leu-Gly

significance

protein are

synthetic

C.

physiological There

the

C, especially

sequence

that

kinase

ganglioside-stimulated to be established.

the

observations

suggest

by protein

kinase

with These

II).

phosphorylate (1 mg/ml).

protein

to a peptide

al.

The

for

could

if

this

corresponding

requirement

also

phosphorylated 18)

can serve

region

whereas

to may

by protein a

synthetic

as a substrate

for

Vol. 165, No. 1, 1989 protein are

kinase

complex

BIOCHEMICAL

C (15).

(Figs.

determination

of

required results

to

presented

ganglioside-stimulated myelin

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

primary for

better

protein

effects

1 and 2; Tables the

of phosphorylation basic

The

and

of

gangliosides

I and II).

sequences

the

the

protein

protein

roles

report

suggest

kinase

of that

may be

studies

including

1 and 2, the preferred

structure-function

functional this

on MBP phosphorylation

Further

of sites

ganglioside-stimulated

understand in

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

kinase,

and site

relationship gangliosides.

3 are

of myelin Nevertheless,

phosphorylation important

the sites

in

of

MBP by

regulating

some

functions.

and Moscarello, M.A. (1978) Biochim. Biophys. Acta. 515, lBoggs, J.M., -21. Benjamins, J.A., and Morell, P. (1978) Neurochem. Res. 3, 137-147. Poduslo, J.F., and Braun, P.E. (1976) J. Biol. Chem. 250, 1099-1105. Kirschner, D.A., and Ganser, A.L. (1980) Nature 283, 207-210. Mikoshiba, K., Kohsaka, S., Takamatsu, K., and Tsukada, Y. (1981) Brain Res. 204 , 455-460. Omlin, F-X., Webster, H. deF., Palkovits, C.G., and Cohen, S.R. (1982) J. Cell Biol. 95, 242-248. ---Schwab, V.S., Clark, H.B., Agrawal, D., and Agrawal, H.C. (1985) JNeurochem. 45, 559-571. DesJardins, K.C., and Morell, P. (1983) J. Cell Biol. 97, 438-446. Murray, N., and Steck, A.J. (1983) -J. Neurochem. 41, 543-548. Murray, N., and Steck, A.J. (1984) J. Neurochem. 43, 243-248. Carnegie, P.R., Kemp, B-E., and Dunkley, P.R. (1973) Biochem. J. 135, 569-572. Miyamoto, E., and Kakiuchi, S (1974) J. Biol. Chem. 249, 2769-2777. Turner, R.S., Chou, C.-H.J., Mazzer, G.J., Dembure, P., and Kuo, J.F. (1984) J- Neurochem. 43, 1257-1264. Kishimoto, A., Nishiyama, K., Nakanishi, H., Uratsuji, Y., Nomura, H., Y. (1985) L-L-' J Biol. Chem 260 12492-12499. Takeyama, Y., and Nishizuka, Chan, K.-F.J., Stoner, G.L., Hashim, G.A., and Huang, K.-P. (1986) Biochem. Biophys. Res 2d-r Commun 144 1287-1295. Ulmer, J.B., and Braun, P.E. (1986) Dev. Biol. 117, 502-510. Chan, K.-F.J. (1987) _--J. Biol. Chem. 262, 2415-2422. Moscarello, M.A., Stoner, G.L., and Webster, H.deF. Chan. K.-F.J.. (1987) Biochem. Biophys. Res L&-I Commun 144 1287-1295. Schulz, P., Crux, T.F., and Moscarello, M.A. (1988) Biochemistry 3, 7793-7799. Martenson, R.E., Law, M.J., and Deibler, G.E. (1983) J. Biol. Chem. 258, 930-937. Chan, K.-F.J. (1987) ---J. Biol. Chem. 262, 5248-5255. Huang, K.-P., Chan, K.-F.J., Singh, T.J., Nakabayashi, H., and Huang, F.L. (1986) --J. Biol. Chem. 261, 2134-2140. Svennerholm, L. (1964) J. Res. 5, 145-162. - Lipid

100