Phosphorylation of fodrin (nonerythroid spectrin) by the purified insulin receptor kinase

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 127, No. 2, 1985

Pages 493-500

March 15, 1985

PHOSPHORYLATION OF FODRIN (NONERYTHROID SPECTRIN) BY THE PURIFIED INSULIN RECEPTOR KINASE a b* a Takashi Kadowaki , Eisuke Nishida , Masato Kasuga c . a . b Tetsu Akivama , Fumzmaro Takaku , Masaharu Ishlkawa , Hikoichi Sakai b, Satish Kathuria d, and Yoko Fujita-Yamaguchi d aThe Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-i Hongo, Tokyo, Japan 113 b

Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, 7-3-i Hongo, Tokyo, Japan 113

CMeiji College d Department

Received January

of Pharmacy,

1-35-23

Nozawa,

Tokyo,

Japan

of Molecular Genetics, City of Hope Research Duarte, California 91010

28,

154 Institute,

1985

SUMMARY: Fodrin (nonerythroid spectrin) from porcine brain was found to be phosphorylated on tyrosine residues b~ the purified insulin receptor kinase. The phosphorylation occurred in an insulin-sensitive manner with a physiologically relevant Km. The ~ (235 K) subunit of fodrin, but not the (240 K) subunit, was phosphorylated by the kinase. Neither the ~ (240 K) subunit nor the ~ (220 K) subunit of erythrocyte spectrin was phosphorylated under the same conditions. Fodrin phosphorylation by the purified insulin receptor kinase was markedly inhibited by F-actin. These data raise the possibility that tyrosine phosphorylation of fodrin plays some roles in the regulation of plasma membrane-microfilament interaction. ©1985Acad~iePress. Inc.

Tyrosine-specific retroviral polypeptide

transforming

proteins

growth factcrs

derived growth factor (7,8).

protein kinase activity

is important because and phenotypes

(i) and of some cellular receptors

such as epidermal

(3), insulin

Identification

of cellular

substrate proteins

they may be involved

proteins

growth factor

(4-6), and insulin-like

in normal and malignant

Cytoskeletal

is a property of many for

(2), plateletgrowth factor-I

for tyrosine

kinases

in the control of cell growth

cells.

are thought to participate

shape and motility but also in cell metabolism

not only in cell

and growth control.

It has

* To whom correspondence should be addressed. Abbreviations: NaDodS04, sodium dodecyl sulfate; Hepes, 4(2-hydroxyethyl)-1-piperazine ethanesulfonic acid.

0006-291X/85

493

$1.50

Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 127, No. 2, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

been shown that transformation of cells by peptide cytoskeletal of tyrosine the p u r i f i e d

growth

systems kinases

factors

(10-13)

in the cells. with

insulin

proteins.

(MAPs)

spectrin,

(9) and treatment

altered

organization

interactions

We p r e v i o u s l y

found that

native

tubulin

is known

to be composed

of ~

It has been shown that fodrin occurs

in most

the p l a s m a membrane

(15-18).

Thus,

mediating

membrane-microfilament

we report

that the p u r i f i e d

tyrosine

fodrin

phosphorylation

is thought

interactions.

of fodrin

receptor

(240 K)

ubiquitously

and it can cross-link

to be a prime

insulin

and

(14).

and ~ (235 K) subunits. cells underlying

of

us to study

kinase p h o s p h o r y l a t e s

proteins

nonerythroid

induce

This p r o m p t e d

cytoskeletal

receptor

microtubule-associated Fodrin,

of cells by retroviruses

candidate

F-actin

for proteins

In this communication, kinase

can catalyze

the

in vitro.

MATERIALS AND METHODS: Insulin receptor was p u r i f i e d 2500-fold to near h o m o g e n e i t y from Triton X-100 solubilized human placental membranes by sequential affinity chromatography on wheat germ a g g l u t i n i n - S e p h a r o s e and insulin-Sepharose, as described p r e v i o u s l y (19). Fodrin was isolated from porcine brains by the m e t h o d of Davis and Bennett (20) with a modification. Briefly, fodrin was extracted at low ionic strength, and then p u r i f i e d by ammonium sulfate precipitation, gel filtration on Sepharose CL-4B, and p h o s p h o c e l l u l o s e chromatography. Spectrin, was extracted from bovine erythrocyte ghosts and p u r i f i e d by gel filtration on Sepharose CL-4B (21). R a b b i t skeletal muscle actin was p r e p a r e d by the m e t h o d of Spudich and Watt (22), and further p u r i f i e d by gel filtration on Sephadex G-100 as p r e v i o u s l y described (23). [y_32p] ATP was from N e w E n g l a n d Nuclear. Porcine insulin was from Eli Lilly Co.. Purified insulin receptor kinase (i-5 pl of a solution c o n t a i n i n g 0.1-0.5 pg of protein) was incubated with or w i t h o u t 10-7M insulin for 45 m i n at 25°C and pH 7.4 in 60 D1 of the standard p h o s p h o r y l a t i o n m i x t u r e containing 40 mM Hepes, 0.i % Triton X-100, MnCI 2 (3 mM), and MgCl 2 (i0 mM). To this solution i-I0 ~g of fodrin or erythrocyte spectrin was added and incubated for 30 sec. Then, p h o s p h o r y l a t i o n reaction was i n i t i a t e d by adding i00 ~M ~ 32p] ATP (10-20 ~Ci/nmol). After incubation for i0 to 180 min at 25°C, the reaction was t e r m i n a t e d by adding u n l a b e l e d ATP (Sigma) to a final c o n c e n t r a t i o n of 2 m M and 30 ~i of 5-fold c o n c e n t r a t e d Laemmli sample buffer (24) containing 100 mM dithiothreitol, then b o i l i n g this m i x t u r e for 3 min. The samples were analyzed by N a D o d S O 4 / p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s followed by autoradiography. P h o s p h o a m i n o acids were identified as d e s c r i b e d p r e v i o u s l y (6). The p h o s p h o r y l a t e d p r o t e i n s were eluted from the gels by e l e c t r o p h o r e s i s and subjected to acid h y d r o l y s i s in 6N HCI for 1.5 h at ll0°C. The p h o s p h o a m i n o acids were resolved by electrophoresis at pH 3.5 followed by autoradiography.

494

VoI. 127, NO. 2, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESULTS Incubation revealed

one

of t h e p u r i f i e d

32p-labeled

Incubation

of t h i s

of

ATP

[~_32p]

incorporated was

into

(6).

spectrin

(5 Vg)

(10-7M)

revealed

in t h e s e examine

that there

preparations the action

(Fig.

purified

incubated

in the p r e s e n c e

ATP.

insulin

Under

B

C

D

E

FODRIN

F

This

~-subunit

fodrin

(5 ~g)

o f M n 2+ a n d M g 2+ f o l l o w e d conditions,

H

I

we

insulin

of

kinase

insulin activity

G a n d H).

on purified

and each of the

32p

or erythrocyte

C a n d D, kinase

of

addition

phosphoprotein

or presence

lanes

i, lane A).

before

o f the

i, l a n e s receptor

[ y_ 32p ] A T P

(Fig.

insulin

o r no e n d o g e n o u s

receptor

these

G

to the

with

in the a m o u n t

in t h e a b s e n c e

COOMASSIE BLUE INSULIN RECEPTOR (+) (-) (+) (-} c+) INSULIN (-) (+) (-) (+) (-) (+) (-) (+) (-) (+) 10-TM

A

10-7M

is l i t t l e

of insulin

spectrin,

[~_32p]

ATP

with

l, l a n e B).

of purified

[~_32p]

kinase

an M r = 9 5 , 0 0 0

increase

(Fig.

to c o r r e s p o n d

with

with

a 3- to 4- f o l d

Incubation

receptor

preparation

this protein

shown previously

receptor

polypeptide

receptor

caused

insulin

To

fodrin

substrates

and

were

by the addition

found phosphorylation

of

o f the

32p-AUTORADIOGRAPHY (+} M.W

J

SPECTRIN

(-)

(+)

A

B

(-3

(+>

(-) (+) (-)

C

D

E

(-)

(+)

(-) (+) (-) (+)

F

G

FODRIN

Fig. I. Phosphorylation of fodrin by the purified insulin receptor kinase. Porcine brain fodrin (5 ~g, lanes C-F) or bovine erythrocyte spectrin (5 ~g, lanes G-J) was incubated with 0 . 1 H g of insulin receptor kinase in the presence or absence of 10-7M insulin. Other assay conditions were described under "MATERIALS AND METHODS". The reaction products were analyzed by NaDodSO4-5 % polyacrylamide gel electrophoresis followed by autoradiography. Direct counting of each subunit band of fodrin excised from the gel cofirmed preferential phosphorylation of ~ (235 K) subunit (-insulin, ~ (240 K) 26 cpm, (235 K) 156 cpm; +insulin, ~ 37 cpm, ~ 798 cpm).

495

(+3

H

I

SPECTRIN

Vol. 127, No. 2, 1985

(235 K)

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

subunit,

lane E).

but not the ~

Insulin

(10-7M)

stimulated

of fodrin by a p p r o x i m a t e l y the ~

(240 K)

subunit

was p h o s p h o r y l a t e d (I0-7M)

was detected

by insulin (Fig.

receptor

i, lanes

little

for 180 min.

kinase

These

phosphorylates

same assay conditions

in the absence

phosphorylation

phosphorylation

reaction

purified

were almost Since

kinase

kinase

insulin Several and

under the

kinase

revealed

exclusively

is known

Insulin

in the presence

to interact

for substrates

(10-TM)

stimulated

change

of

the

in affinity

(Fig.

2).

by

of insulin

(10-7M)

(15-18),

it appears

3). with actin

phosphorylation

to addition

This

that were p h o s p h o r y l a t e d

(Fig.

of F-actin.

prior

was i n c o r p o r a t e d

that the Km of fodrin

in the presence

to know whether

a

for 180 m i n in the p r e s e n c e

reported

in fodrin

tyrosine

reached

kinase was 0.4 ~M fodrin.

the Vmax with little

kinase

receptor

1.7 ~g of fodrin was p h o s p h o r y l a t e d

analysis receptor

30 m i n

although

0.20 mol of p h o s p h a t e

(25-27).

receptor

fodrin with F - a c t i n

F-actin

~-actinin,

for at least

of the insulin

receptor

acid residues

fodrin

that purified

10-7M insulin,

insulin

Kinetic

to be of importance altered

of

When

by increasing

insulin

alone was

as used for the

such as vinculin,

is lower than those p r e v i o u s l y

The amino

of insulin

to cross-link

receptor

neither

spectrin

form of fodrin molecule.

5-10 min.

by insulin

receptor

conditions

of fodrin was linear

and I00 ZM ATP,

per mol of fodrin.

insulin

When fodrin

by insulin

or p r e s e n c e

by 0.5 ~g of p u r i f i e d of 10-7M insulin

of erythrocyte

I and J).

proteins

of the ~-subunit

level w i t h i n

In contrast,

irrespective

data indicate

i,

(data not shown).

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

Km value

subunit

(Fig.

of ~ subunit

kinase

native

filamin were not p h o s p h o r y l a t e d

maximal

I, lane F).

or no loss of the ability

other m i c r o f i l a m e n t - r e l a t e d

either

(Fig.

at 25°C under the same solution

kinase reaction,

receptor

5-fold

of fodrin

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

nor the ~ (220 K)

pretreatment

inct~ated

(240 K) subunit,

As shown

in Fig.

of p u r i f i e d

496

of fodrin

could be

4, incubation

insulin

receptor

of

Vol. 127, No. 2, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

P-Ser P-Thr

12 I1 1Q

P-Tyr o/

7

E

--

/ 5

/

~

withIgsulin

origin Fig. 2. Lineweaver-Burk plot of the phosphorylation of fodrin by the purified insulin receptor kinase. The initial velocity of phosphorylation of fodrin was estimated during a 30 min incubation in the presence (-- :) or absence (o---o) of 10-7M insulin as described under "MATERIALS AND METHODS". The following concentrations (ZM) of fodrin were used: 0.15, 0.2, 0.3, 0.45, 0.6. The molecular weight of fodrin was assumed to be 475 K (~+B). Fig. 3. Phosphoamino acids analysis of phosphorylated fodrin. 30 ~g of fodrin was phosphorylated by 0.5 ~g of insulin receptor kinase for 180 min at 25°C in the presence of 10-7M insulin. Phosphoamino acids analysis was performed as described under "MATERIALS AND METHODS".

kinase

caused

a marked

in t h e p r e s e n c e a n d C)

of

In contrast,

on phosphorylation

to interact

in the phosphorylation

4, l a n e s B a n d D)

10-7M insulin.

by F-actin known

(Fig.

reduction

and absence only

specifically

with

actin

(Fig.

a slight

of exogenously

added

(Fig.

4,

of fodrin

both

4, l a n e s A

effect

was observed

casein which

is n o t

l a n e s E - H).

DISCUSSION The

~ (235 K)

phosphorylated

on tyrosine

The phosphorylation physiologically

subunit

of purified residues

occurred

relevant

Km.

native

fodrin

by purified

insulin

in an insulin-stimulated In view

of that both

497

was

found to be receptor

manner

fodrin

with

kinase. a

and insulin

Vol. 127, No. 2, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3Zp-AUTORADIOGRAPHY CASEIN

(-)

ACTIN

(+)

(-)

INSULIN (10-7M)

(+)

(_) -

(+) (_) (+) ~

(_)

(+)

(_) (+)

(_) (+)

~

<-- FODRIN •<- INSULIN RECEPTOR

waeltle

ACTIN CASEIN

A

B

C

D

E

F

G

H

Fig. 4. Effects of F-actin on phosphorylation of fodrin by the purified insulin receptor kinase. Fodrin (5 ~g) was preineubated at 25°C for 15 min with (lanes C and D, lanes G and H) or without (lanes A and B, lanes E and F) F-actin (15 ~g) in the standard mixture in which the enzyme and ATP were omitted. The reaction was then initiated by the addition of purified insulin receptor kinase and ~ 32p] ATP, and incubations were carried out at 25°C for 30 min in the presence (lanes A-D) and absence (lanes E-H) of casein (30 Ug)- The reaction products were analyzed by NaDodSO4-12 % polyacrylamide gel electrophoresis followed by autoradiography. The result of direct counting of each protein band excised from the gel was as follows: lane E, fodrin 233 cpm, casein 3212 cpm; lane F, fodrin 510 cpm, casein 7524 cpm; lane G, fodrin 38 cpm, casein 2562 cpm; lane H, fodrin 75 cpm, casein 5910 cpm.

r e c e p t o r k i n a s e are l o c a t e d at the inner s u r f a c e of p l a s m a m e m b r a n e , it is r e a s o n a b l e to e x p e c t t h a t this f o d r i n p h o s p h o r y l a t i o n b y the i n s u l i n r e c e p t o r k i n a s e r e a l l y o c c u r s in vivo.

Indeed, p r e l i m i n a r y

e x p e r i m e n t s h a v e s u g g e s t e d t h a t the ~ (235 K) s u b u n i t of f o d r i n e x i s t s as a p h o s p h o p r o t e i n in c u l t u r e d c e l l s . * * * It has b e e n r e p o r t e d t h a t t r a n s f o r m a t i o n of c e l l s b y r e t r o v i r u s e s and t r e a t m e n t of c e l l s by e p i d e r m a l g r o w t h f a c t o r growth factor

(11,12), a n d i n s u l i n

(i0), p l a t e l e t - d e r i v e d

(13) can i n f l u e n c e g r e a t l y the

o r g a n i z a t i o n of a c t i n f i l a m e n t a n d a c t i n - m e m b r a n e i n t e r a c t i o n .

Since

r e t r o v i r a l t r a n s f o r m i n g p r o t e i n s and r e c e p t o r s of t h e s e p e p t i d e g r o w t h

,w,

T. K a d o w a k i , M. K a s u g a ;

E. N i s h i d a , T. A k i y a m a , H. Sakai, F. Takaku, Unpublished observation.

498

and

Vol. 127, No. 2, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

factors are tyrosine kinases,

it is tempting to speculate that the

effect of transformation and growth factors on the structure

of actin

is caused by tyrosine phosphorylation of proteins involved in the regulation of actin polymerization,

actin filament cross-linking

and/or actin-membrane interaction.

In this sense, marked inhibition

of fodrin phosphorylation in the Presence of F-actin is of great interest. Whether tyrosine phosphorylation of fodrin by several tyrosine kinases could alter its function is currently investigated in our laboratory.

ACKNOWLEDGMENT This work has been supported by grant (82R 273) from the Juvenile Diabetes Foundation International to M.K., grants (57440004, 5878016) from the Ministry of Education, Science and Culture of Japan to E.N. and H.S., and grants (AM 20770,AM 34427) from National Institutes of Health to Y.F-Y. We are grateful to M. Kagami and Y. Fujita for their technical assistance.

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