Analyses of phosphorylated tryptic peptide of spectrin from human erythrocyte membrane

Vol. 79, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ANALYSES OF PHOSPHORYLATED TRYPTIC PEPTIDE OF SPECTRIN FROM HUMAN ERYTHROCYTE MEMBRANE* James L. Wyatt, Cancer

Research

Received

Alfred

Institute,

November

C. Greenquist,

University

and Stephen

of California,

B. Shohet

San Francisco,

CA, 94143

15,1977

Summary: Labelling of the membrane protein spectrin with 32P in intact human erythrocytes involves a limited and specific reaction. Sephadex G-55 chromatography and fingerprint analysis of the tryptic digest of [ Plspectrin reveals the presence of a single labelled peptide. Principal phosphate modification takes place at a serine residue (90%) but a small amount of phosphothreonine (10%) is also present. Protein for

kinase

dependent

posttranscriptional

activity

.

protein

modification

Recently

many studies

phosphorylation

in membrane

events

transport

proteins phies cell

(5,7) (5).

and exhibits A number

(111,

hereditary

disease

(2,9)

phosphorylation One of the protein spectrin

kinase

principle

membrane

and phosphatase

paper

of cell were

in

protein

shape

(2,5,6)

dystroand sickle

of some component

polypeptides intact

which

red blood

is of interest

to evaluate

*This work was presented at the 61st Biological Chemists in Chicago, Ill,,

Copyrighl 0 1977 by Academic Press, Inc. All righfs of reproduction in any form reserved.

muscular

of the

membranes.

and deformability

undertaken

phosphatase

of membrane

including

of a disruption

in erythrocyte

This

and protein

and stomatocytosis

mechanism

(5,10,14).

in the control in this

show evidence

(8)

states

spherocytosis

may have significance

phosphorylation

of pathological

associated

(12).

kinase

reversible

one mechanism

and enzyme

membrane

reactions

functions

protein

is

structure

have identified and these

has both

of proteins

of protein

or structural

The erythrocyte activities

phosphorylation

cells because

. the

is a substrate is Band 2 of of its

The studies

specificity

for

possible reported

of the

Meeting of the American in April, 1977 (171.

role

phos-

Society

of

1279 ISSN 0006-291X

BIOCHEMICAL

Vol. 79, No. 4, 1977

phorylation

reaction

present

in spectrin

suggest

that

only

by examining from whole a limited

substrate

for

the protein

Materials

and Methods:

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the number cells

portion

incubated

of labelled with

of the spectrin

tryptic

32Pi' molecule

peptides

The results serves

as a

kinase.

Labelling of Intact Human Red Blood Cells: Blood, freshly drawn into was diluted to a hematocrit of 10% with isotonic saline heparinized tubes, and passed over a cellulose column to remove white cells (1). The red cells were collected and washed three times with isotonic saline. The washed cells were suspended to a final hematocrit of 10% in an isotonic solution , pH 7.4, containing 50 mM imidazole buffer, 5 mM adenosine, 3.1 mM glucose, 25 units/ml of penicillin and 250 mcg/ml of streptomycin, and 100 pCi/ml of 82 Pi. The solution was incubated for 20 hours at At the end of this period, the red cells were washed three 37" (5). times with normal saline and ghosts were prepared by hypotonic lysis (3). Low ionic strength extract containing spectrin was prepared by diluting the membranes 1 to 10 with 0.1 mM EDTA, pH 8; and incubating the suspension for 20 minutes at 37'. The suspension was then centrifuged at 48,200 x g for 60 minutes. The supernatent was drawn off carefully, concentrated by ultrafiltration to 2-3 mg/ml of protein, and frozen. Low molecular weight material was removed from the labelled extract by passage over a G-50 Sephadex column (1.5 x 75 cm) equilibrated with 14.5 mM mercaptoethanol XIdm: r;;ir bicarbg? ate, pH 7.8, containing The [ Pjspectrin emerging in the void volumn was pooled and lyophilized. Sodium Dodecylsulfate-Pol.yacrylamide Gel Electrophoresis (SDS-PAGE): Samples of membrane extract were electrophoresed according to the procedure of Weber and Osborn (15) on 3% and 5% acrylamide gels, Gels were stained for protein with Coomassie Brilliant Blue and were sliced and counted for radioactivity . Alternatively, the gels were sliced longitudinally and autoradiographed (4). _. ' Tryptic-Digestion of [32P]Spectrin: Samples containing 2 to 3 mg of labelled protein were lyophilized. The dried samples were dissolved in 5 mM dithiothreitol 0.5 ml 50-mM ammonium bicarbonate, pH 7.8, containing and 8 M urea. After standing at room temperature for 3 hours the sample was diluted 1:4 with 50 mM ammonium bicarbonate, pH 7.8, containing 5 mM dithiothreitol. TPCK-trypsin was added in two aliquots to give a final spectrin:trypsin ratio of 1O:l. Half the enzyme was added initially and the rest after three hours, The reaction was carried out for 20 hours at 37" under toluene and was terminated by lyophilization. Sephadex G-50 Chromatography of Tryptic-Pkptides: The dried tryptic digest was dissolved in 0.5 ml 10% acetic acid. The sample was then placed on a Sephadex G-50 (medium) column (1.5 x 78 cm) previously equi ibrated in 10% acetic acid. Fractions were collected and monitored for $2 P by measuring Cerenkov radiation, The radioactive peak was pooled and lyophilized. Fingerprint Analysis: The radioactive peak isolated from Sephadex G-50 chromatography was dissolved in a minimal volume of 10% acetic acid (20004000 CPM/pi). An aliquot (l-2 ul) was placed on a TLC-cellulose plate wetted with 5% formic acid, pH 1.9, or pyridine acetate buffer, pH 3.5, The electrophoresis was continued for 2.5 hours at 24 volts/cm. The plate was air dried overnight and then chromatographed at a right angle to the electrophoresis using one of the following solvent systems; butanol:acetic acid:water (4:2:4), the upper phase of the butanol:acetic acid:water (4:1:5)

1280

Vol. 79, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

or pyridine:butanol:acetic acid:water (15:10:1:4). The plate was air dried and overlaid with a sheet(s) of Kodak No-Screen X-ray film for varying times, Determination of Phosphorylated Amino Acids: Samples were prepared for analyses of labelled amino acids by acid hydrolysis or protfylytic digestion. Acid hydrolysis was carried out by dissolving the [ Plspectrin The tubes were evacuated and samples in 1 ml 2N HCl in hydrolysis tubes. After hydrolysis, samples were dried under heated to 110" for varying times. vacuum. Proteolytic digestion was effected by treating a [32P]spectrin sample with a series of enzymes. In a typical experiment, 1 mg of labelled protein was incubated with 50 ug of the protease or peptidase for 24 hours The enzymes used were at 37" in 50 mM ammonium bicarbonate, pH 7.8. carboxypeptidase A and B, and leucine aminopeptidase, TPCK-trypsin, pronase, respectively. Digestion was terminated by lyophilization. Samples were dissolved in a minimal volume of 10% acetic acid A 2 ~1 aliquot was spotted on a TLC-cellulose plate (1000-1500 CPM/pi). and electrophoresed using formic acid:acetic acid: water (1:4:45), pH 2.2, The plate was air dried and autoradiographed for 4 hours at 20 volts/cm. using Kodak No-Screen X-ray film, The autoradiogram was photographed and scanned using a Joyce-Loebl densiometer. Results

and Discussion:

Labelling with

32Pi

From

isolated

sites

led

of Human Erythrocytes: to phosphorylation erythrocyte

of membrane

Several

other

The label during

radioactive

associated

low ionic

Although

trace

Tryptic

about

involved were

and Gel Filtration

in 10% acetic

acid.

column.

A single

85% of the radioactivity

by cyanogen of 8740,

weight

by comparing

2780,

bromide

Fig.

applied of 4800 the elution cleavage

with

from the membrane

0.1 mM EDTA (Fig.

label

were

was found

present

2 illustrates

Tryptic

for

of equine

and 1780).

1281

with

the labelled

volume

after

peptides column

the 32P elution peak (Peak

was eluted

of this

1B).

in Band 2 of spectrin.

a G-50 medium Sephadex

symmetrical

1).

in the 5% SDS-PAGE.

Chromatography:

over

(Fig.

Band 2 and 3 of spectrin.

was removed

weight

proteins

the principle

observable

of the ghost

molecular

were passed

molecular

also

both

Band 2 of spectrin

complex

was determined

weights

labelling

erythrocytes

bound

can be seen that

95% of the radioactivity

Digestion

from this

An estimated

prepared

it

extraction

of intact

membrane

membranes

of lower

over

of the spectrin equilibrated

with

amounts

of several

regions

strength

3% SDS-PAGE gel,

profile

protein

Incubation

A) containing

a K,,

of 0.26.

tryptic

peptide

peptide

to markers

cytochrome

c (molecular

Vol. 79, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMfJNlCATlONS

A Fig,

1:

(A) 5% SDS-PAGE of for protein with graphed (right). strength extract autoradiographed

erythrocyte membranes stained 32 P-labelled Coomassie Brilliant ue (left) or autoradio(B) 3% SDS-PAGE of "P-1abelled low ionic of membranes stained for proteins (left) or (right).

Volume

Fig.

2:

Eluote

(ml)

Gel filtration chromatogaraphy of trypsinized [32P]-spectrin on A 0.5 ml aliquot of the tryptic digest Sephadex G-50 fjqedium). of "desalted [ PI-spectrin" was applied to a Sephadex G-50 column (1.5 x 75 cm> and eluted ith 10% acetic acid. The 1 ml fractions were assayed for Y2 P by monitoring the Cerenkov radiation.

1282

Vol. 79, No. 4, 1977

When

spectrin

matographed with

on

shorter

isolated

was a G-50

times and

of

Peak

of

graphic

and

electrophoretic

spectrin

Peak

of

Table

I summarizes

periods it 90%

is

Peak

the the

was

was

chro-

obtained

when

Peak

was

produced

as

A was after

the

limit

tryptic

To

analyse

the

digestion

the

twenty-four is

in

although

the

only

one

phosphate

residues

might

account

resolved

until

the

either by

both

to

for

modified presented

this. peptide here

case

methods

or

of by

the

It tryptic

electrophoretic

digestion. extrapolating obtained Peak

presented

acid

phosphorylated,

is

interesting

peptide

However

this

question

is

isolated

and spectrin

A,

for

in

Table

I i.e.,

that

fraction

opposing

the

proteolysis.

data

component closely

erythrocyte

when

peptide,

amino

1283

to

previously

phosphothreonine

principle

that

it

transferred

2 N HCl

obtained

From

suggest

a

and

results

in

the

tryptic

between

these

been

be by

were

same

Materials

phosphate

has

to

10% phosphothreonine.

persists

involving

the

of

chromato-

phosphorylated

found

hours.

(see

3)

cell. It

radioactive

(Fig.

From

the

are

samples

composition

other

used

case.

intact

obtained

of

serine

each

hydrolyzed

treated

Several

also

Acid:

also

samples to

spot,

threonine

results

and

results

Amino

phosphoserine

phosphothreonine

The

A,

the

of

phosphoserine

equilibrium

was

then

autoradiogram

contains

in

and

acid

that

phosphoserine

peak

The

in

peptide

ratio

two

obvious

new

were

noted

This

the

for

from

was

serine (13).

peptide,

hydrolyzing

and

profile

Peptides:

conditions

Phosphorylated

both

time

Tryptic

phosphorylation

tryptic

by

A represents

a single

tryptic

proteins

zero

Peak

only

one

membrane

to

no

A shows

only

during

values

trypsin,

prepared.

spot

that

trypsin

Alternatively

were

one

reported

with

elution

maps

on1.y

Analysis

The

.

of

fingerprint

that

same

incubation

Analysis

and

hours

spectrin.

map

appears

the

with

peptide

Methods)

48

for

Therefore, [32P]

A,

digested

redigested

Fingerprint of

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

column,

of

rechromatography. product

BIOCHEMICAL

containing is

seryl cannot

observed. and be

An threonyl

fully

sequenced. modification

by

BIOCHEMICAL

Vol. 79, No. 4, 1977

Fig,

3:

AND BIOPHYSICAL RESEARCH COMMLlNlCATlONS

The peptide map was Autoradiogram of a peptide map of Peak A. prepared by electrophoresing an aliquot of Peak A (2000-4000 CPM) on a thin layer cellulose plate wetted with pyridine acetate, pH 3.5, for 2.5 hours at 24 volts/cm. After drying, the plate was chromatographed at a right angle using butanol:acetic acid: water (4:2:4). The dried TLE plate was overlaid with Kodak No-Screen x-ray film and developed for 18 hours.

TABLE Analysis

of

the

Phosphorylated

I

Amino

Acids,

Phosphorylated

Amino

Serine Acid

Hydrolysis*

Proteolysis

Acid

(%>

Threonine

85

15

90

10

[32P]spectrin was hydrolyzed by acid hydrolysis or proteolysis and analyzed by electrophoresis on thin layer cellulose plates. The plates were autoradiographed and photographed. The relative amounts of phosphoserine and phosphothreonine were determined by densitometric scans using a Joyceebl densitometer. [ 4s Plspectrin *Values are obtained by hydrolyzing for varying times and extrapolating the ratio of phosphoserine to phosphothreonine to zero time,

phosphorylation to

is

determine

membrane

if bound

the

a limited sequence

polypeptide,

and around spectrin,

specific

reaction.

the

It

phosphorylation will

1284

exhibit

the

will

be

interesting

site

in

the

same

features

Vol. 79, No. 4, 1977

described is

currently

for

the

BIOCHEMICAL

soluble

underway

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

substrates

in our

of protein

kinases

(16).

This

work

laboratory.

The authors wish to acknowledge the excellent technical Acknowledgements: assistance of Mrs. Mary Rossi. This work was supported by USPHS Grant AM16095 from the National Institutes of Health. References: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16, 17.

Beutler, E., Red Cell Metabolism: A Manual of Biochemical Methods, 2nd ed,, (1975), p. 10, Grune and Stratton, New York. Beutler, E., Guinto,E., and Johnson, C. (19761, Blood, 68, 887. Dodge, J.T., Mitchell, C., and Hanahan, D.J. (19631, Arch. Biochem. Biophys., 100, 119. Fairbanks, G., Levinthal, C., and Reeder, R.H. (19651, Biochem. Biophys. Res. Comm,, 20, 393, S.B. (1975), Erythrocyte Structure and Greenquist, A.C., and Shohet, p. 515, Alan R. Liss, New York, Function, S.B. (1976), Blood, 48, 877. Greenquist, A.C., and Shohet, Graham, C., Avruch, J,, and Fairbanks, G. (19761, xochem. Biophys. Res. comm., 72, 701. Guthrow, C.E., Allen, J.F., and Rasmussen, H. (19721, J. Sic& Chem., 247, 8145. Hosey, M., and Tao, M. (19761, Nature, 263, 424. Palmer, F.B., and Verpoorte, J.A. (1971), Can. .J. Biochem., 9, 337, Roses, A.D., and Appel, S.H. (1975), J. Membrane Biol., 20, 51. Rosen, O.M., and Rubin, C.S. (1975), Ann. Rev. Biochem., 44, 831. Biochem, Biophys. Res. Comm., Rubin, C.S., and Rosen, C.S. (19731, lo, 421. V. (1977), J. Biol. Chem., 252, 508. Shapiro, D., and Marchesi, Chem., 244, 4406. Weber, K., and Osborn, M, (19691, J. Biol. Williams, R.E. (19761, Science, 192, 473. Wyatt, J.L., Greenquist, A.C., and Shohet, S.B. (1977), Fed. Proc, 2, 640.

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