Phosphorylation of P36 in vitro by protein kinase C

vol. 137, No. 1, 1986

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

May 29, 1986

Pages

PHOSPHORYLATION Navin

C.Khannna+,

Masaki

Department Received

The

OF P36 IN VITRO Tokuda+,

of Medical Calgary,

April

11,

Sarah

BY PROTEIN M. Chong,

KINASE

397-403

C

and David

M. Waisman*

Biochemistry University of Calgary Alberta T2N 4Nl CANADA

1986

subunit of protein I (~36) is a major substrate of Here we demonstrate that protein tyrosine protein kinases. kinase C catalyzes the incorporation of 1.7 moles of phosphate per mole of protein I. Phosphorylation is absolutely dependent on the presence of both calcium and phospholipid, and is specific for serine and threonine residues. Phosphorylation of protein I by the c-AMP dependent protein kinase, phosphorylase kinase, casein kinase I, and casein kinase II was not observed. The in va significance of protein kinase C dependent phosphorylation of p36 is discussed. 0 1986 Academic Press, Inc. 36kDa

several

A 36,OOOkDa

protein,

called

in

Rous

phosphoprotein

the

and in EGF stimulated the

36kDa

that

the

protein

from

they named proteinI. of

two copies

Protein

of

porcine

(3).

both

of

Gerke

intestinal

to

of

1OkDa subunit

be immunologically

related

protein

(4) isolated and showed protein

which

(Mr 85,000). to

the

tyrosineprotein kinase

(1,2)

atetramerconsisting

and

tyrosine

cells

and Weber

a larger

of Rous sarcomavirus-encoded

and the EGF stimulated

as the major

epithelium

I was showntobe a 36kDa

identified transformed

virus

was a subunit Protein

I was shown

phosphoprotein

sarcoma

A431 cells

protein

36kDa

p36 has been

36kDa kinase

of the A431cells

*To whom all correspondence should be addressed. +Postdoctoral fellows of the Alberta Heritage Foundation Medical Research. Supported by a grant from the Medical Council of Canada. Abbreviations: EGF, Epidermal Growth Factor; SDS-PAGE, Sodium Sulphate Polyacrylamide Gel Electrophoresis; DTT,Dithioerythritol; Carboxy-methyl; DEAE, Diethylaminoethyl; EGTA, Ethylene glycol ( -aminoethyl ether)-N,N,N',N-Tetraacetic acid.

For Research

Dodecyl CM, bis-

0006-291X/86

397

AN

Copyright I? 1986 righrs of reproduct/on

(5).

$1.50

by Amdemic Press. Inc. i?r crp. fbrm rewrvrd.

Vol. 137, No. 1, 1986 P36 has been of protein

BIOCHEMICAL

shown to exist

I but

Recently

also

Sawyer

in

presence the

and Cohen

both

protein.

authors

Since

concluded of for

been of

protein

kinase

the the

C.

I

is

This

data

of of

EGF

p36 from

and demonstrated

phosphoserine

residues

(6)

that

suggested

receptor/kinase residues.

serine

presents protein

vitro

the

possibility C may

kinase

of

communication,

kinase

the

The protein

as an in

p36 these

catalyzed

phosphorylation

present

p36 in --

as a component

EGF receptor/kinase

identified data

activation

phosphorylation

and

purified

the

only

A431 cells

p36 on tyrosine

In

not

immunoprecipitated

previous

EGF stimulated

protein

EGF dependent

Materials

of

identified.

subunit

serine

the

that

phosphorylation

never

have

phosphotyrosine

substrate

responsible

(3)

RESEARCH COMMUNICATIONS

(5).

Pi-labelled,

of

was &I vitro

in A431 cells

as a monomer

EGF stimulated,intact, the

AND BIOPHYSICAL

p36 has

the

36kDa

substrate

of

that

the

result

in

the

vivo.

and Methods

Calcium and phospholipid-dependent protein kinase C was purified from bovine brain as described (7). Protein I (36K and 1OK) was purified from bovine lung according to the procedure of Shaddle et al (5). [Y-~~PI ATP (10 Ci/mmol) was purchased from New England Nuclear. DEAE-cellulose (DE-52) and CM-cellulose was obtained Phenyl-sepharose-4Bwas frompharmacia. 1,2-diolein, fromwhatman. L-a-phosphotidyl-L-serine, diisopropylflurophosphate @I-), soyabean trypsin inhibitor, phosphoserine, phosphothreonine, and phosphotyrosine wereobtained fromsigma ChemicalCompany. Reagents for protein determination and gelelectrophoresis were obtained from Bio-Rad . Calcineurin was a generous gift from Dr. J.H.Wang (University of Calgary). Casein kinase I, casein kitale I;i;;f phosphorylase kinase were generous gifts from Dr. . . (University of Calgary). Calcium-activated phospholipid-dependent protein kinase was Piincorporation from[y-3 P]ATP intoprotein I assayed by measuring as described by (7). The assay was carried out in a total volume of 200~1 containing 25mM Tris-HCl, pH7.5, 1OmM magnesium chloride, 0.5mM DTT, 1OOug L-a-phosphatidyl-L-serine, 1Oug diolein, 12.5uM 600uM calcium chloride, and enzyme [Y-~~PIATP (1400cpm/pmol~f, Aliquots phosphotidyl serine (5mg/ml) and protein. diolein (5mg/ml) were mixed in chloroform and evaporated under The residues were resuspended in 20mM Tris-HCl, nitrogen gas. PH7.5, and sonicated for a total time of 5 minutes on ice with a Branson sonicator. Phosphorylation reaction was initiated by the addition of[y-32P]ATP. Incubations were carried out at 30°C for various time intervals up to two hours. Reaction was terminated by the addition of lml of 25% trichloroacetic acid containing 2% The acid precipitated material was collected sodiumpyrophosphate.

Vol. 137, No. 1, 1986

8lOCHEMlCAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

on Whatman 3mm filterpaper funnel and washed five times with 4mls of 5% TCAcontainingl% sodiumpyrophosphate. 10 mls of scinti-verse was added to the filters and the radioactivity determined. Control experiments with enzyme protein alone in the absence of either/or calcium and phospholipid were also carried out by including 1mM EGTA (in the absence pf calcium chloride) or replacing the phospholipid mixture with 20mM Tris-HCl, pH7.5, containing 1OmM Phosphorylase kinase activity was assayed at magnesium chloride. 20mM Tris, pH7.5 containing 1OmM magnesium chloride and O.lmM calcium chloride. Endogenous phosphorylating activity of all kinases was determined in the absence of substrate and served as blanks. To evaluate the effect of calcineurin or alkaline phosphatase on the calcium and phospholipid-dependent phosphorylation of ~36, the reaction was terminated by addition of 2mM EGTA to remove the calcium. Calcineurin (5119) or alkaline phosphatase (Spg) was added to the reaction mixture and incubation continued for another 30 minutes. Aliquots were removed at 5 and 30 minutes for SDS-PAGE. SDS-PAGE was carriedoutaccording totheprocedure of Laemmli (8). Coomasie Blue was used to visualize the protein bands on the gel. For autoradiography the stained gel was dried on a filter paper to X-Omatic AR film in a Kodak with a gel dryer and then exposed X-Omatic cassette at -7O'C. Phosphoaminoacidanalysiswasperformed accordingtotheprocedure of Wong et al (9). Gel bands containing the phosphorylated 36kDa subunit were cut from the stained gel. The p36 was electroeluted and dialyzed against water to remove the electrode buffer. To the dialysate, 5Oug of bovine serum albumin was added and the protein was precipitated with 20% TCA. After centrifugation the protein pellet was washed first with ice cold ethanol and then with ethanol, The washed pellet was ether mixture (l:l, v/v). hydrolyzed with 6N HCl for 3 hours at 110 C. The solvent was evaporated using The hydrolysate was a Savant speed vaccum. resuspended in 60~1 of water and an aliquot was applied to 20 x 20 cm precoated cellulose thin-layer plates. A solution containing phosphoserine, phosphotyrosine and phosphothreonine (5OmM each, 2~1) was spotted with the sample. Electrophoresis was carried 1ooov using pyridine/acetic acid/water, out at pH3.5 at dried and exposed 1:10:189 (v/v). The plate was then for autoradiography. After autoradiography the plate was sprayed with ninhydrin reagent and baked at 100 C for 5 minutes to allow visualization of the phosphoamino acid standards. Results Protein Shaddle

I

was

purified

et al

(5).

The

(~36)

36kDa

and

1OkDa

theobservationsof of

the

the

(data

Incubation analysis

not

SDS-PAGE

of the

SDS-PAGE

bovine

lung

according

to

protein

gave

two

of

(figure

a

therefore 36kDa

I (36k

1).

bands

Consistent

with

(4) gelpermeationchromatography

yields

shown),

of protein by

on

protein

form

monomeric

purified

Gerke and Weber

purified

Mr 85,000

from

single

protein

peak

of

the

absence

of

suggesting

subunit. and 10k)

autoradiography 399

with

protein suggest

kinase that

C and Pi

was

Vol. 137, No. 1, 1986

BIOCHEMICAL

PAGE

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Autorodioaroph

94 67 43 30

20 14

+ +

+ -

-

- co

+

-PL

Figure 1. One-dimensional SDS polyacrylamide gel (left) and autoradiography (right) of the ~36 preparation (20 Dg/ml) after b vitro phosphorylation by protein kinase. The numbers indicate the mobilities of molecular mass standards in kilodaltons (kDa): phosphorylase b (94) ; bovine serum albumin (67); ovalbumin (43); carbonic anhydrase (30) : soyabean trypsin inhibitor (20.1) : a-lactalbumin (14.4). directly

incorporated protein

I

(figure

dependent

protein

were

(figure

kinase

85,000

the

for

alkaline

of

p36 (figure In

order

protein

Figure

1).

the tetramer

or

to

was

1.7

(51,

2 presents

resulted

in

identify

the

phosphoamino

phosphorylated

by

cellulose

sheet.

The autoradiograph

was detected

Inadditiontoprotein other

~36.

rapid

of

the

Assuming

Mr

protein

I

of calcineurin dephosphorylation

of

C and

by chromatography is

C, protein

kinases.

residues

kinase

presented

on phosphoserine

kinase protein

acid

protein

6 N HCl and analyzed

several

course

and

inset).

with

for

calcium

a time of

the

the

of p36 was totally

The addition

hydrolyzed

Radioactivity

of

moles of phosphate/m01

60 minutes.

phosphatase

subunit

both

of

phosphorylation

after

2,

36kDa

presence

C catalyzed

incorporated

the

The phosphorylation

1 inset).

on

phospholipid

into

in

p36 the

partially on thin figure

3.

and phosphothreonine.

I wastestedasa

As shown in table

substrate 1 the c-AMP

BIOCHEMICAL

Vol. 137, No. 1, 1986

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

P-Thr

3Or---P-Tyr

0

02

30

60

0 120 90 MINUTES

150

Id0

origin

03

Figure 2. Time course of phosphorylation and dephosphorylation of ~36. Purifiedp36preparationwasphosphorylatedbyproteinkinase C under various conditions. Aliquots were taken at different time intervals for liquid scintilation spectrophotometry and for autoradioqraphy. Calcineurin (20ug/ml) alkaline phosphatase (20pg/ml) was added at thearrow and reaz:ioncontinued. Inset: Shows the autoradiograph of SDS-PAGE of the time course reaction of phosphorylation and dephosphorylation. Time course is as follows: l', 2', 5', lo', 20', 30', 60', 120', 125', 150'. The arrow indicates the addition of Calcineurin or Alkaline Phosphatase. 0 in the presence of Calcium (0.6mM) and Phospholipid (see text) 0 in the absence of Calcium and Phospholipid A Calcineurin 0 Alkaline Phosphatase Figure 3. Phosphoamino acid analysis of the phosphorylated ~36. Portions containing ~36 were cut from the SDS-PAGEand electroeluted with 2% Agarose gel. Partial acid hydrolysis was performed and phosphoamino acids were seperated on a cellulose plate by electrophoresis (lOOOV, 60min) at pIi3.5 (pyridine/acetic acid/water, 1:10:189) as described under "Materials and Methods*. Marker phosphoamino acids are phosphoserine (P-Ser), phosphothreonine (P-Thr) and phosphotyrosine (P-Tyr), identified by ninhydrin staining. Autoradiography with an intensifying screen was carried out on a Kodak X-omatic AR film. dependentproteinkinase, or casein of

kinase

labelled

subunit

II

phosphorylase (11) did not catalyze

phosphate

of protein

kinase,

into

either

the

caseinkinase

significant 36kDa subunit

I

(10)

incorporation or

the

1OkDa

I.

Discussion The results kinase

presented

C catalyzes

the

in this

communication

stoichometric 401

suggest

phosphorylation

that of

protein the

36kDa

Vol. 137, No. 1, 1986

BIOCHEMICAL

Table 1 Phosphorylation

of Protein

Kinase

mole

Protein

Casein Kinase II

0.002

of

Kinase

presence

both

serine

I,

0.01

&

of both

vitro.

calcium

and threonine

impressive

body

calcium

of

information

activation

major

phosphorylated

serves

kinase

on both

demonstration

1) ,

of

p36 it

is

is

EGF

responsible

for

the phosphorylation

residues.

The significance

by protein

kinase

vitro

For example,

autophosphorylation

autophosphorylation in vitro

unclear

serine

site site

with

for

the

the report

(5)

~36, in

substrate to

postulate and

the in VW 402

in

serine

site

p36

vs &I has 3 ti

1 comparing

will

of

vitro

EGF receptor/kinase

studies

C

and serine

reflect

only

EGF

kinase

but

Further

protein

protein

phosphorylation

but

and

that

threonine might

is

vivo, of

of p36 on tyrosine

the

target

messenger

residues

vitro

sites (14).

cytosolic

EGF receptor/kinase,

receptor/kinase

of

suggesting

receptor-linked

Considering

reasonable

the

differences.

to

in their

from

an b

of

viya

localized

elicit

formation

and tyrosine

activation

C is

on

accumulated

as a second

the

serine

that

dependent

and is

as EGF,

C (12,131.

substrate

C (figure

such

derived

turnover

of protein

the

has

and diacylglycerol

Diacylglycerol

phosphoinositide

is

and phospholipid

signals,

mobilization

cells.

Phosphorylation

residues.

many extracellular

kinase

0.003

Kinase

protein

the

the

protein”

Assuming a molecular. weight of 85kDa

subunit

An

Kinases

1.7 0.006

a.

Protein

P;incorporation/mole

Casein Kinase I

Phosphorylase

that

RESEARCH COMMUNICATIONS

I by Various

Kinase C

CAMP-dependent Protein

that

AND BIOPHYSICAL

in-

vivo the p36

be neccessary

BIOCHEMICAL

Vol. 137, No. 1, 1986

to

unequivocally

kinase

establish

C phosphorylation

the

AND BIOPHYSICAL

ih

vd

RESEARCH COMMUNICATIONS

significance

of

protein

of ~36.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Padke, K., and Martin , G.S. (1979) Proc, Natl. Acad. Sci USA 76, 5212-5216 Erikson, R.L., Colett , M.S., Erikson, E., Purchino, A.F., and Brugge, J.S.(1979 1 Cold Spring Harbor Sym. Quant. Biol. 44, 907-917. Sawyer, S.T., and Cohen, S. (1985) J. Biol. Chem. 260, 8233-8236. Gerke, V., and Weber, K. (1984) Embo. J. 3, 227-233. Shaddle, P.J., Gerke, V., and Weber, K. (1985) J. Biol. Chem. 260, 16364-16360. Fava, R.A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645. Kikikawa, U., Takai, Minakuchi, R., Inohara, S. and Nishizuka, Y. (1982). J. Biol. Chem. 257, 133431-13348. Laemmli, U.K. (1970) Nature 227, 680-685. Wong, T.W., and Goldberg, A.R. (1983) J. Biol. Chem. 258, 1022-1025. Singh, T.J., and Huang, K.P. (1985) FEBS Lett. 190, 84-88. Huang, K.P., Itarte, E., Singh, T.J., and Akatsuka, A. (1982) J. Biol. Chem. 257, 3236-3234. Takai, Y., Kaibuchi, K., Tsuda, T., and Hoshijima, M. (1985) J. Biol. Biochem. 29, 143-155. Beridge, M.J. (1984) Biochem. J. 220, 345-360. Dawnward, J., Waterfield, M.D., and Parker, P.J. (1985) J. Biol. Chem. 260, 14538-14546

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