Detection of a novel lymphocyte protein-tyrosine kinase by renaturation in polyacrylamide gels

Vol. 134, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

January 29, 1986

Pages 963-969

Detection of a Novel Lymphocyte Protein-Tyrosine Kinase by Renaturation in Polyacry]amide Gels Robert L. Geahlen and Marietta L. Harrison Department of Medicinal Chemistry and Pharmacognosy School of Pharmacy and Pharmacal Sciences Purdue University, West Lafayette, Indiana 47907 Received November 26, 1985

Summary-Protein kinase a c t i v i t y , including a c t i v i t y specific for the phosphory l a t i o n of tyrosine residues, can be detected among p a r t i c u l a t e fraction proteins of T cell lymphomas a f t e r separation by SDS-polyacrylamide gel electrophoresis. Putative protein kinases are detected by renaturation of enzyme a c t i v i t y d i r e c t l y within the gel following removal of detergent. LSTRA, a cell line that exhibits elevated levels of p r o t e i n - t y r o s i n e kinase a c t i v i t y , was found to express a predominant p r o t e i n - t y r o s i n e kinase of molecular weight 30,000. This same enzyme was present in T lymphocytes and other T lymphoid Cell l i n e s . Studies involving rapid preparation of protein f r a c t i o n s , limited proteolysis and one-dimensional peptide mapping did not demonstate a direct relationship between the phosphorylated 30,000 dalton protein and the predominant 56,000 dalton phosphotyrosine containing protein that is observed following phosphorylation of LSTRA cell p a r t i c u l a t e fractions in v i t r o . ® 1986Academic Press, Inc.

Protein-tyrosine cell

growth

based

kinases have been implicated as important regulators of on t h e i r i d e n t i f i c a t i o n

as viral

growth factor receptors ( f o r reviews, see 1,2). tic

cells,

high

oncogene

products

Among nontransformed eukaryo-

lymphocytes of both T and B lineages have been found

levels of p r o t e i n - t y r o s i n e kinase a c t i v i t y

and

(3-5).

to

express

The best studied

of

the lymphocyte p r o t e i n - t y r o s i n e kinases is an enzyme from T c e l l s . Much of the information which

regarding this enzyme comes from studies of the cell

possesses

(7-9).

line

highly elevated levels of p r o t e i n - t y r o s i n e kinase

LSTRA, activity

The enzyme from LSTRA is a protein of 56,000 daltons that catalyzes an

autophosphorylation reaction that modifies a tyrosine residue ( I 0 ) . We have been developing rapid methods for the detection and tion

of protein kinases in crude,

SDS-polyacrylamide of

kinase

activity

identifica-

heterogenous protein samples by

combining

gel electrophoresis with in situ (in the gel) renaturation (ii).

Using this procedure to

examine

protein

kinases

0006-291X/86 $1.50 963

Copyright © 1986 by Academ~ Press, Inc. A# r~h~ of reproduction m any form reserveK

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present

fractions from LSTRA c e l l s ,

in

endogenous

particulate

protein-tyrosine

kinase a c t i v i t y ,

we have i d e n t i f i e d

but one that has

an

an

apparent

molecular weight on SDS-polyacrylamide gels of 30,000.

Materials and Methods Cell lines were cultured as described previously (9,12). Particulate fractions w e r e prepared from c e l l s and endogenous protein substrates were phosphorylated as described (4). Peptide mapping of labeled protein bands excised from polyacrylamide gels was performed according to Cleveland et a l . (13). For phosphoamino acid analyses, labeled proteins were recovered from dried polyacrylamide gels (14) and hydrolyzed for I h at IIO°C in 5.7 N HCI. Samples were l y o p h i l i z e d , dissolved in H20 , and electrophoresed on Whatman c e l l u l o s e t h i n - l a y e r plates at pH 3.5 (5% acetic acid, 0.5% pyridine) for 50 min at i i 0 0 V in the presence of standards of phosphoserine, phosphothreonine, and phosphotyrosine (Sigma). Protein kinases w e r e detected in SDS-polyacrylamide gels as follows: Mixtures of proteins were s o l u b i ] i z e d by heating at 90°C for 4 min in an SDS sample buffer consisting of 2.5% SDS, 25% sucrose, 25 mM Tris/HCl, pH 8.0, 2.5 mM EDTA, 1.5% d i t h i o t h r e i t o l , 2.5 mg% pyronin Y. Proteins were separated on SDS-polyacrylamide slab gels (Hoeffer model SE600) consisting of a 4% polyacrylamide stacking gel and a 9% running gel. Bovine serum albumin at i0 pg/ml was included in the running gel solution p r i o r to i n i t i a t i o n of polymerization. Following electrophoresis, gels were washed extensively in 40 mM Hepes, pH 7.4, for 6 hours to remove SDS (15). Protein kinases were detected by incubation in 50 ml of 25 mM Hepes, pH 7.4, containing 10 mM MnCI9 and 500 pCi [?-~CP]ATP (3000 Ci/mmole, Amersham or >7000 Ci/mmol, New England~Nuclear) f o r 3 h at room temperature. Excess unincorporated ATP was removed by an overnight incubation in 600 ml of 40 mM Hepes, pH 7.4, containing 20 g Dowex 2X8-50 anion exchange resin. Gels were then incubated for i h in 5% ( v / v ) acetic acid, 10% (v/v) isopropanol, 1% (w/v) sodium pyrophosphate. Proteins were stained with Coomassie B r i l l i a n t blue and labeled proteins were detected by autoradiography of the dried gels. Results and Discussion Detection of protein kinases in LSTRA c e l l s - A number of d i f f e r e n t enzymes have been

renatured successfully following electrophoresis

gels

(15-19).

protein

on

SDS-polyacrylamide

We have recently adapted these procedures to the detection

kinase a c t i v i t y (11),

The r e l a t i v e migration positions

of the kin-

ases are determined by phosphotransferase assays carried out d i r e c t l y the gel m a t r i x . kinases was

electrophoresis

within

We have applied in s i t u renaturation to the study of protein

present in the cell l i n e LSTRA.

prepared

of

A postnuclear p a r t i c u l a t e

from

LSTRA c e l l s and separated

into

on

an SDS-polyacrylamide gel.

Following removal of

described in Materials and Methods,

protein

fraction

components

by

SDS as

the gel was incubated with [~-32p]ATP and

MnCl 2 to allow pnosphotransferase reactions to occur. 964

As shown in Fig. 1 A, a

Vol. 134, No. 2, 1986

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A

B

92 66

45

31

1

2

Figure 1. Detection of protein kinases in particulate fractions from LSTRA cells. ~, samples of particulate fraction protein containing 0.8 mg (lane 1) or 0.4 mg (lane 2) of protein were fractionated by electrophoresis on a 9% SDS-polyacrylamide gel. The gel was washed to remove SDS and to allow enzymes to renature withi~2the gel matrix. The gel was then incubated with 10 mMMnCl and 500 NCi [?-~ P]ATP (3000 Ci/mmol). The migration positions of protei~ kinases were determined by autoradiography of the fixed, stained and dried gel. Numbers on the l e f t refer to the molecu|ar weights (in KDa) of standard proteins of known size. B, the phosphorylated protein band migrating at an apparent molecular weight of 30,000, identified in Fig. I, was extracted from the polyacrylamide gel and hydrolyzed for 1 h at 110% in 5.7 M HCI. Phosphoamino acids were separated by e|ectrophoresis on cellulose thin-layer plates, detected by autoradiography, and identified by co-migration with authentic standards of phosphoserine (P-ser), phosphothreonine (P-thr), and phosphotyrosine (P-tyr). number of d i s t i n c t phosphorylated proteins could be observed. radiolabeled

bands had apparent molecular weights,

The f i v e

calculated

by

major

relative

migration p o s i t i o n , of 30,000, 42,000, 54,000, 62,000 and 80,000. The

three most intensely labeled protein bands were labeled to an extent

sufficient The

54,000

to allow ready i d e n t i f i c a t i o n of the phosphorylated

amino acids.

Da protein was found to be phosphorylated on a threonine

residue

and the 42,000 Da protein on serine (data not shown). S u r p r i s i n g l y , the 30,000 Da protein (p30) was labeled e x c l u s i v e l y on tyrosine (Fig. tein

i B).

Since pro-

kinase a c t i v i t y was detected subsequent to SDS-polyacrylamide gel

elec-

trophoresis, the monomer molecular weight of the active subunit of t h i s enzyme must be 30,000 Da. 965

Vol. 134, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

Y

L

B

YAC-1

30

I

Figure 2. Protein kinases from YAC-I and LSTRA c e l l s . Particulate fractions were prepared from YAC-I (Y) or LSTRA (L) c e l l s . A, protein samples (20 Pg) were phosphorylated prior to gel electrophoresis as described in Materials and Methods. Phosphoproteins were separated by SDS-gel electrophoresis and detected by autoradiography. The migration position of p56 is indicated. B, particulate fraction proteins prepared from YAC-I cells ( i mg, lane i ; 0.5 mg, lane 2) were separated by SDS-polyacrylamide gel electrophoresis. Protein kinases were then renat~ed within the gel and detected by incubation with 10 mM MnCI9 and 500 pCi [~- P]ATP (7000 Ci/mmol). The migration position of the 30,000 Ba protein is indicated. Comparison late

of p56 and p30- The major substrate observed when a

f r a c t i o n prepared from LSTRA c e l l s is phospnorylated in v i t r o

particuprior

to

SDS-po|yacrylamide gel e l e c t r o p h o r e s i s is a phosphotyrosine containing protein of Mr = 56,000 (p56) (7 and Fig. 2A). S u b c e l l u l a r f r a c t i o n s from T lymphocytes and other T lymphocyte-derived c e l l

l i n e s (such as YAC-I, which is shown here)

also catalyze the phosphorylation of p56 (9 and Fig. 2A). To tions

determine i f p30 could be detected in YAC-I c e l l s ,

particulate frac-

were prepared and separated by SDS-polyacrylamide gel

electrophoresis.

Protein kinases were renatured and detected by incubation with [~-32p]ATP. number of enzymes were v i s i b l e in the f r a c t i o n from YAC-I c e l l s including (Fig.

A p30

2B). S i m i l a r r e s u l t s were found f o r p a r t i c u l a t e f r a c t i o n s prepared from

the spontaneous leukemic c e l l l i n e SL-2, and thymocytes (not shown). 966

and from normal spleen T lymphocytes

Vol. 134, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

42 B 30

0

5

10

15

20

40

60

p30-

Figure ~. Comparison of p30 and p56. A, LSTRA c e l l s (3 X 106 ) were pelleted by centrifugation at 12,000 x g for i0 sec and immediately placed in SDS-sample buffer and heated in a boiling water bath for 5 min. Proteins were then separated by electrophoresis on a 10% SDS-polyacrylamide gel. Protein kinases ~ r e detected by renaturation and incubation with I0 mM MnCI~ and 500 ~Ci [~ P]ATP (7000 Ci/mmol). The migration positions of phospho~rote~ns migrating at M = 30,000 and 42,000 are indicated. B, LSTRAc e l l s ( i X i0 ) were incubate~ on ice with 2 ml of 5 mM Hepes, pH 7.4, I% t r i t o n X-IO0 and were broken in a Dounce homogenizer. Soluble proteins were incubated at 30°C for the times indicated (in minutes). Samples (I0 ~I) were phosphorylated and separated by SDS-polyacrylamide gel electrophoresis as described in Materials and Methods to detect p56. A l t e r n a t i v e l y , samples (I00 ~I) were fractionated by gel electrophoresis and p30 was detected by in situ renaturation.

There

is much evidence to suggest t h a t p56 is a p r o t e i n - t y r o s i n e

(9,10).

Since

many p r o t e i n - t y r o s i n e kinases can be degraded to a c t i v e 30,000

Da subunits (20-22), we were i n t e r e s t e d in the p o s s i b i l i t y arisen due to a p r o t e o l y t i c used

kinase

d i g e s t i o n of p56.

three experimental approaches,

t h a t p30 might have

To examine t h i s

question,

we

i ) Protein samples were r a p i d l y prepared

by b o i l i n g i n t a c t LSTRA c e l l s in SDS-sample b u f f e r and p30 was detected by

in i

situ

renaturation (Fig.

3A).

From t h i s

result,

it

appeared u n l i k e l y t h a t the

30,000 Da enzyme o r i g i n a t e d from a l a r g e r precursor unless conversion occurred p r i o r to the d i s r u p t i o n of the c e l l s .

2) T r i t o n X-IO0 e x t r a c t s of LSTRA c e l l s

were incubated at 30°C f o r v a r y i n g periods of time. autophosphorylation reactions, change

While p56, as detected by

was degraded w i t h t i m e ,

there was no apparent

in the amount of p30 detected by in s i t u r e n a t u r a t i o n ( F i g . 967

3B).

3)

Vol. 134, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

B

z

mr

0

I

2

3

4

,5

6

7

8

MIGRATION DISTANCE (cm)

Figure 4. One-dimensional peptide mapping of p56 and p30 from LSTRA c e l l s . The p30 protein, labeled as described in Fig. 1A, and p56, phosphorylated as described in Fig. 2A, were excised from polyacrylamide gels and re-electrophoresed on a second 15% SDS-polyacrylamide gel in the presence of 0.375 ~ug of Staphylococcus aureus V8 protease. Phosphopeptides were detected by autoradiography. A, densitometric scan of phosphopeptides generated from p56. --B, densitomeEric scan of phosphopeptides generated from p30.

One dimensional peptide maps of phosphorylated p56 and p30 did not reveal apparent s i m i l a r i t i e s observations,

it

between the two proteins (Fig.

4). Based on these three

is u n l i k e l y t h a t p30 o r i g i n a t e d from p56.

that p30 o r i g i n a t e d from a d i f f e r e n t ,

any

The

possibility

as yet uncharacterized p r o t e i n - t y r o s i n e

kinase, however, can not be ruled out. The r e s u l t s of t h i s study demonstrate the u t i l i t y for

the i d e n t i f i c a t i o n

t h i s procedure,

of in s i t u renaturation

of protein kinases in crude protein

fractions.

Using

we have i d e n t i f i e d a novel p r o t e i n - t y r o s i n e kinase present in

T lymphoid c e i l s with a molecular weight of 30,000. The o r i g i n s of t h i s enzyme and i t s r e l a t i o n s h i p to other known p r o t e i n - t y r o s i n e kinases, to be determined. activity

and

Further work w i l l

however, remain

be necessary to c h a r a c t e r i z e t h i s

to determine the role that t h i s and other kinases play

enzyme in

the

complex i n t e r a c t i o n s t h a t r e s u l t in the a c t i v a t i o n of lymphocytes.

Acknowledgments This i n v e s t i g a t i o n was supported by PHS grants R01 CA37372 and P01 CA36761 awarded by the National Cancer I n s t i t u t e , DHHS. We thank Jonathan Scher and P h i l i p Votaw f o r t h e i r technical assistance. 968

Vol. 134, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS References

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