The Regulatory Role of Known Tyrosine Autophosphorylation Sites

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 2, Issue of January 15, pp. 990-996, 1991 Printed in U.S.A.

The Regulatory Role of Known Tyrosine Autophosphorylation Sites of the Insulin Receptor Kinase Domain AN ASSESSMENT BY REPLACEMENT WITH NEUTRAL AND NEGATIVELY CHARGED AMINO ACIDS*

(Received for publication, August 6, 1990)

Bei ZhangS, Jeremy M. Tavar%ll, Leland Ellisg, and Richard A. RothII From the Department of Pharmacology, Stanford University School of Medicine, Stanford, California 94305 and the §Howard Hughes Medical Institute and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9050

Autophosphorylation of the insulin receptor has been previously documented to activate the phosphotransferase activity of the receptor from 20- to 200-fold. Biochemical studies have correlated activation of the receptor kinase with the autophosphorylation of tyrosines residues 1158,1162, and 1163. To further assess the role of these 3 tyrosines in the activation process, we have studied the effect of their substitution with either the neutral amino acids phenylalanine or alanine o r with the negatively charged amino acids aspartate and glutamate. In several other proteins, it has been shown that substitution of phosphorylated residues with negatively charged amino acids can mimic the phosphorylation state of the protein. In agreement with previous studies, tyrosines at positions 1162 and 1163 were found to be crucial in the kinase activation process. In contrast, mutant receptors with tyrosine 1158 changed to either phenylalanine or aspartate were still activated to the same extent as the wild-type receptor. An increased basal exogenous kinase activity was observed upon replacement of tyrosines 1162 and 1163 with, in increasing order of potency, aspartate = glutamate < alanine = phenylalanine. These results indicate that phosphorylation of tyrosines 116211 163 but not 1158 play a critical role in the activation of the receptor kinase and that the mechanism of activation of the receptor kinase by autophosphorylation is more complex than just an introduction of a cluster of negative charges in this region of the receptor. In addition, the finding of an increased basal kinase activity in receptors lacking tyrosines 1162 and 1163 could explain the reported ability of this receptor to mediate certain biological responses.

The insulin receptor (IR)’ is a disulfide-linked heterotet-

DK 41765 (to R.A.R.), 34926 (to R.A.R.), and 40511 (to L.E.) and a * This work was supported by National Institutes of Health Grants

grant from the Howard Hughes Medical Institute (to L.E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

4 Supported by a postdoctoral fellowship from the American Dia- betes Association.

1 Supported by a Medical Research Traveling Fellowship. 11 To whom all correspondence should be addressed Dept. of Phar-

macology, Stanford University School of Medicine, Stanford, CA 94305. Tel.: 415-723-5933.

‘The abbreviations used are: IR, insulin receptor; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Hepes, 4- (2-hydroxyethy1)-1-piperazineethanesulfonic acid EGTA, [ethylene- bis(oxyethylenenitrilo)]tetraacetic acid.

rameric membrane glycoprotein consisting of two extracellu- lar a (Mr = 135,000) and two transmembrane p ( M , = 95,000) subunits (for reviews, see Refs. 1-4). Binding of insulin to the a subunit activates a tyrosine-specific phosphotransferase activity in the cytoplasmic domain of the /3 subunit. The receptor then autophosphorylates and, as first shown by Ro- sen et al. ( 5 ) , this autophosphorylation activates the ability of the receptor kinase to phosphorylate various exogenous substrates. Extensive studies of this activation process have documented that: (i) activation occurs in intact cells as well as with purified receptor (6, 7); (ii) it is reversible by phosphatase treatment of the receptor (8); (iii) it is not caused by a change in the quaternary state of the receptor (5) ; (iv) it can occur with fragments of the cytoplasmic domain of the receptor (9, 10); and (v) activation of the receptor kinase correlates best with autophosphorylation of 3 tyrosines (1158, 1162, and 1163)* in the kinase domain of the receptor (11- 20).

Questions still remain, however, as to the relative contri- bution of the 3 tyrosine phosphorylation sites 1158, 1162, and 1163 to the activation of the insulin receptor kinase, as well as to the mechanism whereby phosphorylation activates the receptor kinase. The close proximity of these 3 tyrosine autophosphorylation sites suggests that the introduction of a cluster of negative charges in this region of the kinase domain of the receptor could act to expose the substrate binding portion of the receptor and thereby increase its activity. Support for such a model comes from the finding that peptides with the same sequence as this region of the receptor (as well as other substrates) inhibit both receptor autophosphorylation and subsequent activation of the receptor kinase (21,22). To further test this hypothesis, as well as to assess the role of these 3 tyrosines in the activation of the receptor kinase, we have constructed site-directed mutants of the human insulin receptor in which these 3 tyrosines have been changed to either the neutral amino acids phenylalanine or alanine, or the acidic amino acids aspartate or glutamate. The mimicking of the phosphorylation state of a protein by the introduction of the negatively charged amino acid aspartate by site-directed mutagenesis at the site of a phosphoserine was first demon- strated by Thorsness and Koshland (23) with isocitrate de- hydrogenase. Subsequent studies have also reported successes in mimicking the phosphorylation state of a variety of other proteins by replacing phosphoserines and phosphothreonines with negatively charged amino acids, although this approach has not been reported for phosphotyrosine (24-28). To facil-

sequence of the receptor of Ebina et al. (42). These differ from that The numbering of amino acids in this paper correspond to the

of Ullrich et al. (43) by being 12 higher.

990

Tyrosines 1158, 1162, and 1163 and Insulin Receptor Kinase Activity 99 1

itate the assessment of the affects of these mutations on the basal and activated kinase activities of the insulin receptor, we utilized transient expression in COS cells of the IR cDNAs under the transcriptional control of the SRa promoter, which combines the SV40 early promoter and the R segment and part of the U5 sequences of the long terminal repeat of the human T-cell leukemia virus type 1 (29).

EXPERIMENTAL PROCEDURES

Materials-COS-7 cells were maintained in Dulbecco's modified Eagle's medium with 10% defined and supplemented calf serum (Hyclone), 100 units/ml penicillin, and 100 pg/ml streptomycin (University of California, San Francisco tissue culture facility) a t 37 "C in 5% CO,. Poly(Glu,Tyr)4:1 and Histone F2B were from Sigma. Monoclonal antibodies 5D9 and 2G7 were as described (30). Monoclonal antibody 83-7 and polyclonal antibody Ros-2 were gifts from Dr. K. Siddle (University of Cambridge, Cambridge, United Kingdom) (31). T7-GenTM in uitro mutagenesis kit and SequenaseTM kit were purchased from United States Biochemicals (Cleveland, OH). Other DNA modifying enzymes were from Bethesda Research Labo- ratories (Gaithersburg, MD). [Y-~'P]ATP (6000 Ci/mmol) was purchased from Du Pont-New England Nuclear and [a-%]dATP from Amersham Corp. Porcine insulin was from Elanco. The human insulin receptor cDNA in the pCMVl expression vector (32) was a gift of Dr. J. Pessin.

Site-directed Mutagenesis and Construction of Expression Vectors- Human IR cDNA was subcloned from pECE vector (12) into M13mp19 and used as a single-stranded template for mutagenesis. Oligonucleotide-directed mutagenesis was performed using the T7- GenTM in uitro mutagenesis kit following the manufacturer's instruc- tions. Oligonucleotides were designed to introduce amino acid substitutions in the IR, and wherever possible, restriction enzyme recog- nition sites were either introduced or eliminated in the oligonucleotides to facilitate the screening of mutants. We will use the convention of referring to a mutant receptor with Tyr""' to Asp"" substitution

. The oligonucleotides used and alterations in restriction sites are as follows (the mismatched bases are underlined): Yn58u, 5'-ATCCGTTTCATCGATGTCTCTGG-3' (new ClaI site); Y1158F,

CCCCTTTCCGATCGTCATCCGTTTCATAGATGT-3' (new PuuI

TAGATG-3' (eliminates the PuuI site when Y1162D-Y1163D mutant cDNA was used as template); Y1162A-Y1163A , 5"GCCCCCTTTCC- GACCGCCATCCGTTTCATAGATG-3' (eliminates the PuuI site

mutant cDNA was used as template). To , the mutant cDNA of Y1162D-Y1163D and

the oligonucleotide for Y1lSsD were used. The mutant IR Y1162F-Y1163F was as described (12). The mutations were verified by DNA sequenc- ine and the mutant cDNAs were then subcloned into the SRa

as y l l 5 8 D

5'-ATCCGTTTCAuGATGTCTCTGGTC-3'; Y1162D-Y1163D,5'-GC-

site); y1162E-yI163E ,5"GCCCCCTTTCCGCTCCTCATCCGTTTCA-

when the y116*D-y1163u construct y1158D~y1162D_y1163D

expression vector (29). Exoression of the Mutant ReceDtors in COS-7 and NIH 3T3 Cell

Lines-To transiently express the receptors, subconfluent COS-7 cells grown in a 60-mm Petri dish were transfected with 4 pg of DNA using calcium phosphate co-precipitation as follows: 0.5 ml of a solution containing 4 pg of DNA, 0.124 M CaC12, 39 mM NaCl, 6.8 mM Hepes, pH 7.0, and 0.19 mM NaHP04 was added dropwise to the cells in 5 ml of media. After 18 h a t 37 "C in the incubator, the media was changed and the cells were left for an additional 50-57 h in the incubator. The amount of time and DNA utilized in the transfections were chosen after doing a time course and dose curve, respectively. T o establish cell lines that stably express normal and mutant IR, NIH 3T3 cells ( lo6 cells/60-mm plate) were co-transfected with plasmid DNA (10 pg of each expression plasmid plus 50 ng of pSVneo) using calcium phosphate co-precipitation as described above. After 48 h, Geneticin (400 pg/ml) was added to the culture medium to select for neomycin-resistant cells. Cells expressing high levels of surface IR were identified by '*'I-insulin binding (see below).

SDS-PAGE of IR and Western Blot-COS-7 cells transiently expressing IR were lysed in a buffer containing 50 mM Hepes, pH 7.6, 5 mM EDTA, 5 mM EGTA, 1% Triton X-100,150 mM NaCl, 20 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 20 mM sodium fluoride, and 1 mg/ml bacitra- cin. The cell lysates were mixed with Protein G-agarose beads previously coated with monoclonal antibody 5D9 on a rotator a t 4 "C overnight. The beads were then washed twice with WGBT (50 mM Hepes, pH 7.6, 150 mM NaCl, 0.1% Triton X-100, 0.1% Tween 20,

0.1% bovine serum albumin), and the immunoprecipitated proteins were analyzed with 10% SDS-PAGE. For Western blotting, lysates were loaded directly on 10% SDS-PAGE and the proteins were transferred to nitrocellulose filters after electrophoresis. Protein bands corresponding to IR precursor and the 0 subunit of mature IR were detected with a rabbit polyclonal antibody directed against the isolated kinase domain of the receptor (33). Bound rabbit antibody was detected with an anti-rabbit alkaline phosphatase conjugate and a chromogenic substrate (Promega).

Insulin Binding Assay-Transfected COS-7 cells were plated in a 24-well culture dish 24 h before harvest. The cells were washed twice with ice-cold Hepes-buffered saline, 0.3 mM CaCl,, and incubated with 280 pl/well of buffer A (100 mM Hepes, pH 7.8, 120 mM NaCl, 1.2 mM MgS04, 1 mM EDTA, 15 mM sodium acetate, 10 mM glucose, 1% bovine serum albumin) containing various concentrations of unlabeled insulin a t 4 "C for 1 h. Forty thousand cpm of '251-insulin (-100 pCi/pg) in 20 p1 of buffer A was then added to each well and the incubation was continued a t 4 "C overnight. The cells were washed twice with ice-cold Hepes-buffered saline, 0.3 mM CaCl,, lysed with 0.5 ml of 0.05% SDS, and the radioactivity was counted in a y- counter.

Tyrosine Kinase Assay-Lysates of COS-7 cells transiently expressing IR were prepared as described above. COS-7 cells transfected with SRa vector alone were used as controls in these experiments. Tyrosine kinase activity was assayed using a modified version of the microtiter plate immunoprecipitation method (34). Ninety-six well polyvinylchloride plates (Falcon) were coated with 10 pg/ml of monoclonal antibodies 2G7 or 83-7 against IR in 20 mM NaHC03, pH 9.6 (50 pl/well), and incubated a t 4 "C for 16 h. The wells were then washed three times with WGBT and 40 p1 of the cell lysates were added to each well. The incubation was continued at 4 "C overnight and the wells were washed three times with WGBT. The wells were then treated with 40 p1 of alkaline phosphatase (EC 3.1.3.1., Type VI1 from bovine intestinal mucosa) (Sigma) in 0.1 M Tris, pH 8.0 (0.275 unitlpl), at 25 "C for 15 min followed by three washes with WGBT. To measure the basal tyrosine kinase activity, 20 p1 of kinase reaction mixture (50 mM Hepes, pH 7.6, 150 mM NaCl, 5 mM MgCl,, 5 mM MnCl,, 0.1% Triton X-100, 1 mg/ml poly(Glu,Tyr)(4:1), 2 pCi of carrier-free [y-32P]ATP) was added directly to each well and the incubation was continued a t 25 "C for 3, 6, 9, and 12 min. To determine the maximal activity of the IR kinase, duplicate sets of the dephosphorylated receptor were incubated a t 25 "C for 30 min with 30 pl/well of an activation mixture containing 50 mM Hepes, pH 7.6, 150 mM NaC1, 0.1% Triton X-100, 0.1% bovine serum albumin, 5 mM MgCl,, 5 mM MnC12, M insulin, and 1 mM ATP. The wells were then washed three times with WBGT and the kinase activity was measured as above. The reaction was terminated by spotting 10 p1 of the reaction mixture on a Whatman 3MM filter strip. After air drying, the filter strips were soaked in ice-cold 10% trichloroacetic acid containing 10 mM sodium pyrophosphate (5 ml acid/l X 3-cm strip) for 30 min, boiled in 5% trichloroacetic acid, 10 mM sodium pyrophosphate for 10 min, washed twice with 95% ethanol and once with acetone, and the radioactivity was determined by Cerenkov counting. In each experiment, control wells lacking receptor were assayed and these values (typically 2,000-3,000 cpm) were subtracted. To determine the relative amounts of receptor present in the wells, another set of wells were incubated with 100,000 cpm/40 pllwell of '251-labeled anti-IR monoclonal antibody 29B4 a t 4 "C for 5 h. The wells were then washed three times and counted in a y-counter. In the study of in uiuo activation of IR with insulin, NIH 3T3 cells were washed once with phosphate-buffered saline, incubated with serum-free culture medium for 15 min, and then treated with the indicated concentrations of insulin for 10 min at 37 "C. The cells were then washed with phosphate-buffered saline, lysed as described above for the COS cells, and the tyrosine kinase activity was measured with the microtiter plate assay method without prior treatment with alkaline phosphatase (35).

Tryptic Digestions and Two-dimensional Phosphopeptide Map- ping-Transiently transfected COS-1 cells (confluent 60-mm dish) were extracted into 0.5 ml of ice-cold immunoprecipitation buffer (IPB; 50 mM Hepes, pH 7.5, 0.15 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 2.5 mM benzamidine, 1 pg each of pepstatin, antipain, and leupeptin) supplemented with 1% Triton X-100 and 10 mM EDTA. After 5 min at 0 "C the extracts were clarified by centrifugation a t 10,000 X g for 5 min a t 4 "C. The supernatant was rocked for 2 h a t 4 "C with 5 mg of Protein A-Sepharose (Pharmacia LKB Biotechnology) and 5 pl of rabbit anti-insulin receptor antiserum Ros-2. The immune complex was isolated by brief centrifugation and

992 Tyrosines 1158, 1162, and 1163 and Insulin Receptor Kinase Activity

washed twice with 1 ml of IPB supplemented with 1% Triton X-100 and 10 mM EDTA. The complex was then washed a further two times with 1 ml of IPB with 0.1% Triton X-100 but without EDTA. The pellet was resuspended in 50 pl of 130 mM Hepes, pH 7.5, 12 mM MgCI,, 2 mM MnC12, 100 pg/ml bovine serum albumin, and 1 p~ insulin. After 15 min at 30 "C, autophosphorylation was initiated with 50 p~ [-p:'"P]ATP (2000 cpm/pmol). The reaction was terminated after 10 min at 30 "C with 10 p1 of Laemmli sample buffer and heating to 95 "C for 5 min. The "P-labeled 0-subunit was isolated by SDS- PAGE and digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin as described (19). The resulting phosphopeptides were separated (along with 1 pg of dinitrophenyllysine) on an Eastman Kodak 20 X 20-cm cellulose thin layer chromatography plate by electrophoresis a t pH 3.5 in one-dimension followed by ascending chromatography in the second dimension (19). The plate was dried and autoradiographed at -80 "C using Kodak X-Omat AR film with intensifying screens.

RESULTS

Characterization of the Transiently Expressed Receptor- To test the feasibility of utilizing transient transfections for analyzing mutant insulin receptors, the wild-type human IR cDNA in three different expression vectors (pECE, SRa, and pCMV1) was transfected into COS-7 cells. After 60 h, cells were lysed and the receptor was immunoprecipitated and analyzed by SDS-gel electrophoresis. Coomassie Blue staining of the gel revealed that the highest levels of receptor were obtained with IR in the SRa vector (Fig. LA) and this vector was, therefore, utilized in all subsequent experiments. To verify that the insulin receptor was being expressed in the transient transfections, lysates of control and transfected cells were also analyzed by immunoblotting with an antibody to the kinase domain of the receptor. Considerably higher levels of receptor were observed in lysates of transfected cells (Fig. 1B). Finally, transfected and control cells were compared for their ability to bind I2'I-insulin. The transfected cells were found to maximally bind -120 times (average of three experiments) more insulin than the control cells (Fig. 2). The affinity for insulin of the expressed wild-type and mutant receptors ( K d = 2 nM) were approximately the same as the endogenous receptor ( K d = 1.5 nM).

To assess the kinase activity of the expressed IR, receptors

A

prelR+

I R n +

I R P *

FIG. 1.

a b c d e f B a b

- 4 5

SDS-PAGE and Western blot of IR exmessed in COS-7 cells. A, SDS-PAGE of the expressed receptor: Cells were transfected with vector alone ( b ) , IR cDNA in the SRa expression vector (c), the pECE vector ( d ) , or the pCMVl vector ( e ) . The receptor proteins were immunoprecipitated from lysates of the cells with monoclonal antibody 5D9 and analyzed by SDS-PAGE. Shown is a picture of the Coomassie Blue-stained gel. Lanes a and f contain molecular mass markers with their indicated masses (in kDa). B, Western blot of the expressed receptor. Lysates of COS-7 cells transfected with vector alone ( a ) or the IR cDNA SRa expression vector ( b ) were electrophoresed on SDS-PAGE, transferred to nitrocellulose, and reacted with a polyclonal antibody to the receptor p chain. Indicated are the positions of prestained molecular mass markers (in kDa).

1000

- 1 1 - 1 0 - 9 - 8 - 7 - 6 insulin (log M)

FIG. 2. I2%Labeled insulin binding to COS-7 cells. The binding of '*'I-labeled insulin to COS-7 cells transfected with either wild- type IR cDNA (.) or SRa vector cDNA (A) was measured in the presence of the indicated concentrations of insulin (lZ'II-labeled plus unlabeled). Results are means of triplicate determinations and are representative of three independent experiments.

were isolated on microtiter wells previously coated with monoclonal antireceptor antibodies. To measure the basal kinase activity, the isolated receptors were first dephosphorylated by an incubation with alkaline phosphatase since variable levels of phosphotyrosine were detected on untreated IR (data not shown). The phosphatase was then removed by washing and the basal kinase activity of the receptor on an exogenous substrate, poly(Glu,Tyr)41, was assayed. Autophosphoryla- tion during the assay was minimized by utilizing low concentrations of ATP (30 nM). Parallel wells were assayed for the amount of receptor present by incubation with an iodinated monoclonal antibody to a distinct antigenic site of the receptor. In general, the amount of expressed wild-type and mutant receptors bound to the wells varied by less than 10%. To examine the maximal kinase activity of the autophosphorylated receptor, the phosphatase-treated immobilized receptors were incubated for 30 min at 25 "C with 1 PM insulin and 1 mM ATP in the presence of MgC12 and MnC12. The activation mixture was then washed out and the receptor was assayed for kinase activity on poly(Glu,Tyr)41 as for the basal kinase activity measurements. This activation procedure resulted in an -80-fold increase (average of four experiments) in kinase activity of the wild-type receptor (Fig. 4). This kinase activity was predominantly attributable to the expressed receptor since assays of cells transfected with the wild-type IR cDNA had -110 times (average of six experiments) more kinase activity than COS cells transfected with vector alone.

Introduction of Negatively Charged and Neutral Residues at Tyrosine Phosphorylation Sites-cDNAs encoding mutated receptors containing 1,2, or 3 negatively charged amino acid residues at the 3 tyrosine autophosphorylation sites (1158, 1162, and 1163) were expressed in COS-7 cells and the basal kinase activities of these receptors were compared to the wild- type receptor. Mutant receptor Y1lssD with 1 extra charged residue exhibited -2 times more basal kinase activity than wild-type receptor, whereas mutant Y11G2D-Y11G3D with two extra negative charges was -4 times more active than the wild-type receptor3 (Fig. 3). Mutant receptor Y1162E-Y11G3E had the same basal activity as Y'1G2D-Y11G3D, suggesting that either glutamate or aspartate were equally able to stimulate receptor kinase activity (Fig. 3). Mutant receptor Y'1s8D-Y1162D-Y1163D with three added negative charges had a slightly higher basal activity than mutant Y11G2D-Y11G:3D (Fig. 3).

To test whether the increased basal activity observed in these mutant receptors was specific for the introduction of a

Mutant IRs are referred to with the new amino acid and residue number in the superscript.

Tyrosines 1158, 1162, and 1163 and Insulin Receptor Kinase Activity 993

0 5 1 0

Time (min)

0 5 1 0 1 5

Time (min)

FIG. 3. Basal tyrosine kinase activities of mutated IRs with substitutions of negatively charged residues at Tyr115' and/or Tyr"62-Tyr'163. COS-7 cells were transiently transfected with vector alone (A), SRa expression vector for wild-type receptor (W), or

Y1162D-Y1163D). Receptors from the cells were adsorbed to microtiter wells coated with monoclonal antibody 2G7. The wells were then treated with alkaline phosphatase and the basal tyrosine kinase activity was measured as incorporation of radioactivity into the exogenous substrate poly(Glu,Tyr)4:1. Results are means of triplicate determinations and are representative of three experiments. The amount of the mutant receptors present on the wells (expressed as a fraction of the wild-type receptor) were 0.90 (Y1lsSD), 0.98 (Y1162D-

mutant receptors (A, yllssD; 0, y1162D-y1163D. 0, y1162E.y1163E. ., Y115SD.

y1163D), 1.03 (y1162E.yI163E ), and 1.03 (Y1158D-Y1163D).

negatively charged residue, tyrosine autophosphorylation sites 1162 and 1163 were also changed to either phenylalanine or alanine. The mutant receptors Y1162F-Y1163F and Y1162A-

were both found to have -10 times more basal kinase activity than wild-type receptor, or -2.5 times more basal kinase activity than the receptor which had the same amino acids changed to either of the negatively charged amino acids glutamate or aspartate (Fig. 4A). To verify that another mutation which might be responsible for this activation had not been introduced into the cDNA encoding mutant Y1162F- Y1163F, this cDNA was mutated to encode Y1162D-Y1163D and expressed. This construct yielded a receptor which had the same basal kinase activity as the prior mutant Y1162D-Y1163D.

The effect of these different mutations on the activation of the receptor kinase was also assessed. Mutant IR Y1162F-Y1163F exhibited no significant increase in activity after preincuba- tion with insulin and ATP (Fig. 4B). Similarly, Y1162D-Y1163D

In contrast, mutants Y1158D and Y1'58F showed the same amount of activity after activation as the wild-type receptor (Fig. 5B). These two mutants also exhibited the same activation as wild-type IR when the receptors were immobilized with either a monoclonal antibody to a distinct antigenic site of the receptor or when the receptor kinase was assayed with a different substrate, histone. To see if the kinase activity of mutant Y1162F-Y1163F was affected by tyrosine 1158, the mutant

ity. This mutant was found to have the same basal and activated kinase activities as mutant Y1162F-Y1163F (data not shown).

Further Studies on Mutant Receptors Lacking Autophos- phorylation Site 1158"To examine the in situ activation of the kinase activity of the IR mutant lacking autophosphorylation site 1158, we prepared stable transfectants of NIH 3T3 cells expressing the wild-type IR and mutants Y1158F and

. Intact cells were then treated with different concentrations of insulin, lysed, and the receptor isolated in microtiter wells previously coated with monoclonal anti-in-

y 1 1 6 3 A

and y1162A-yI163A exhibited no significant activation (Fig. 4B).

receptor y1158F-yl162F-yl163F was prepared and tested for activ-

y1162F-yl163F

L 2 150000 I " f h I

2 100000 I a m c p 50000

0 5 Time (min)

1 0

FIG. 4. Tyrosine kinase activities of wild-type and mutant insulin receptors with substitutions at Tyr116Z-Tyr1163. Recep- tors from COS-7 cells transiently expressing wild-type (W), mutant

trol cells (A) were immunoadsorbed to microtiter wells as described. Basal tyrosine kinase activities ( A ) were determined after treating the wells with alkaline phosphatase. Activated receptor kinase activities were determined after incubation with 1 mM ATP and 1 p~ insulin ( B ) . The kinase activity is expressed as incorporation of radioactivity into the exogenous substrate poly(Glu,Tyr)4:1. Results are means of triplicate determinations and are representative of three experiments. The amount of the mutant receptors present on the

receptors (A, y1162D-y1163D; 0, y1162A-y1163A. 0, y1162F-y1163F), and con-

sulin receptor antibodies. Via this technique, we could dem- onstrate that the wild-type receptor exhibited a 25-fold increase in activity with a half-maximal activation at -1 nM insulin (Fig. 6). The mutant receptor Y1158F exhibited the same activation as the wild-type receptor whereas Y1162F-Y1163F was again not activated by insulin.

Tryptic Phosphopeptide Mapping of the Autophosphorylated Wild-type and Y158F Mutant Insulin Receptors-To assess which of the major insulin receptor autophosphorylation sites were phosphorylated in the Y1lssF mutant, COS cells were transfected with either wild-type or Y1158F mutant insulin receptors. Insulin receptors were then isolated from extracts of these cells as immune complexes which were incubated in vitro with insulin and [Y-~'P]ATP. SDS-PAGE performed under reducing conditions revealed the presence of 32P-labeled precursor and mature p subunit in immune complexes of both wild-type and Y1lssF mutant insulin receptor preparations (not shown). Two-dimensional phosphopeptide mapping was performed (Fig. 7).

A key to the identity of the major phosphotyrosine containing tryptic phosphopeptides obtained from the wheat germ lectin-Sepharose purified human placental insulin receptor phosphorylated i n vitro is depicted in Fig. 7e (see also, Ref. 19). In brief, the 3 major tyrosine autophosphorylation sites (kinase domain residues 1158, 1162, and 1163) are recovered as a family of five phosphopeptides (general sequence DI YET- DYYRK), which are mono- (Cl), bis- (B2 and B3), or tris- (A1 and A2) phosphorylated and cleaved by trypsin at arginine 1155 and either arginine 1164 (Cl, B3, and A2) or lysine 1165 (B2 and Al). Tyrosines 1328 and 1334 are recovered as a single bisphosphopeptide (sequence SYEEHIPYTH

Tyrosines 1158, 1162, and 1163 and Insulin Receptor Kinase Activity

A

2000 -

lo00 -

0 0 2 4 6 8 1 0

Time (min)

.""""" I

0 2 4 6 8 1 0

Time (min)

FIG. 5. Tyrosine kinase activities of insulin receptors with substitutions at tyrosine 1158. Lysates of COS-7 cells transiently expressing wild-type (W) or mutant receptors (0, Y I I S R F ; A, Y115R1)), and COS-7 cells transfected with SRa vector alone (A) were prepared and the receptors were adsorbed to microtiter wells coated with monoclonal antibody 2G7. Basal ( A ) and activated ( B ) IRs were assayed for kinase activity as above. Results are means of triplicate determinations and are representative of three experiments. The amount of the mutant receptors present on the wells (expressed as a fraction of the wild-type receptor) were 0.95 (Yll""') and 1.08 (YIISxF).

Insulin (log M)

FIG. 6. In vivo activation by insulin of mutant IR lacking tyrosine 1158. NIH 3T3 parental cells (A), NIH 3T3 cells stably expressing wild-type IR (W), and mutant receptors (A, Y l l R H F ; 0, y l l B ? F ~ y l l l i : l F ) were treated for 10 min with the indicated concentration of insulin. The cells were then washed and lysed and the tyrosine kinase activities of immunoimmobilized receptors were measured. The activities have been plotted as the fold stimulation over the basal activity without insulin treatment for each cell line. Data are means of duplicate assays and are representative of three experiments.

MNGGK) that migrates as peptide B1. Additional tyrosine autophosphorylation sites (probably residues 965, 972, and 984) migrate as peptide Cl', C2, and C3.

As expected, the mature p subunit from the wild-type

a ......, :.......

b

+ - ELECllwFwRESIS d - FIG. 7. Tryptic phosphopeptide mapping of "'P-labeled

wild-type and YIIRRP mutant insulin receptors. Wild-type (a , c) (b , d ) insulin receptors were isolated from transfected COS

cells by immunoprecipitation. Autophosphorylation was performed in the presence of insulin and [y-:"P]ATP and the resulting '"P- labeled insulin receptor mature p subunit (a, b ) or precursor (c, d ) isolated and subject to two-dimensional tryptic phosphopeptide mapping as described under "Experimental Procedures." The drawing ( e ) is a key to the assignment of the major phosphotyrosine containing tryptic peptides derived from the wild-type human insulin receptor phosphorylated in vitro. The arrow and dotted oval represent the point of sample application and position of dinitrophenyllysine, respectively. For further details, see "Discussion."

or y l l A R F

insulin receptor (Fig. 7a) exhibited considerable amounts of the kinase domain trisphosphopeptides A1 and A2 along with peptide B1 from the C terminus. Interestingly, none of the intermediate mono- (Cl) or bis- (B2 and B3) phosphopeptides that lead to the formation of A1 and A2 were detectable suggesting that autophosphorylation had proceeded to com- pletion. An almost identical map was obtained from the precursor (Fig. 7c).

Maps of both mature p subunit and precursor from the

tides B2 and B3 with no detectable A1 or A2 (trisphosphopeptides) or C1 (monophosphopeptide) from the kinase domain. This confirms that tyrosines 1162 and 1163 can be readily autophosphorylated in the absence of tyrosine 1158. Peptide B1 from the C terminus was also observed. Maps of p subunit of the Y115R*' mutant gave essentially identical results to those of the YllsHF mutant (data not shown). The identities of the "'P-labeled material migrating just to the left of the origin in all of the maps shown and just above B1 in Fig. 7d are not known.

y l 1 5 R F receptor (Fig. 7, b and d) exhibited the bisphosphopep-

DISCUSSION

In the present studies we describe the use of transient transfections of COS cells to analyze the affect of mutagenesis of three major autophosphorylation sites of the insulin receptor. This method allows for a more rapid analysis of the affect of these mutations than the previously described use of stable cell lines expressing mutated receptors (12,17). In the present studies we have been able to consistently obtain -100-fold increases in the level of both receptor kinase and insulin binding during these transient transfections. The much greater increase in the present studies compared to a previ-

Tyrosines 1158, 1162, and 1163 and Insulin Receptor Kinase Activity 995

ously reported 4-fold increase in insulin binding in transient transfections of COS cells (36) may in part be due to the use of the strong promoter SRcy and optimization of the transfec- tion protocol.

The expressed receptor appeared to bind insulin with normal affinity and to be processed into the mature cy and p subunits (Figs. 1 and 2). The levels of the precursor receptor are higher than in cells expressing normal levels of receptors but do not differ appreciably from that found in stable cell lines of Chinese hamster ovary cells overexpressing the human insulin receptor (12). This precursor receptor may be capable of transmitting an insulin signal since the phosphopeptide maps of this molecule were identical to that of the mature /3 subunit (Fig. 7). Indeed, using antiphosphotyrosine antibody immunoblotting we have found considerable insulin stimulation of tyrosine phosphorylation of both wild-type and

mutant IR precursors in intact COS cells (not shown). The basal and maximal kinase activities of the wild-type and mutant IRs expressed in these cells could be analyzed after isolation of the receptors on microtiter wells coated with monoclonal antireceptor antibodies. Via this approach, we could obtain an -80-fold activation of the wild-type receptor kinase as a result of autophosphorylation in the presence of insulin (Figs. 4 and 5), a value close to the 20- to 80-fold activation of receptor kinase observed in intact cells treated with insulin (Fig. 6 and Refs. 6, 7, and 35). The lower activation in some of the in vivo experiments may in part be due to the presence of partially tyrosine phosphorylated and activated receptors in the non-insulin treated cells (35). The validity of the current approach was in part verified by the finding that mutant receptor Y11fi2F-Y1163F, which was previously found in stable transfectants not to have any insulin- activated receptor kinase (12), was again found to have essentially the same activity with or without autophosphorylation (Fig. 4).

In the present studies these same 2 tyrosine autophosphorylation sites were also changed to alanine, aspartate, or glutamate. All three new mutant IRs were not significantly activated by autophosphorylation in the presence of insulin (Fig. 4). These results further substantiate the critical role of tyrosines 1162 and 1163 in the activation of the receptor kinase. The basal kinase activities of the two mutant receptors with either of the negatively charged amino acids at these tvo sites were -4-fold elevated over the wild-type receptor. How- ever, the mutant IRs with these 2 amino acids changed to either phenylalanine or alanine exhibited an even higher basal kinase activity than the mutant IRs with the 2 negatively charged amino acids at these residues (Fig. 4). This is in agreement with the previously observed increase in basal autophosphorylation of the mutant Y1162F-Y1163F overexpressed in stable transfectants in Chinese hamster ovary cells in the autophosphorylation reaction (12). The finding that

ave an approximately 10-fold higher basal kinase activity than the wild- type receptor could explain the reported abilities of these mutant receptors to mediate various biological responses (37- 39). It is possible that the substitutions of the tyrosines with these other amino acids affects the conformation of the receptor such that it increases the access of substrates to the active site. The inability of the substituted negatively charged amino acids to further increase the basal kinase activity of the receptor over that observed with either phenylalanine or alanine would suggest that the mechanisms of activation of the receptor kinase by autophosphorylation is more complex than just an introduction of a cluster of negative charges in this region of the receptor. The inability of negatively charged

y l l 5 8 F

mutant IRs y1162F-yI163F and y1158F-y1162F~y1163F h

amino acids to substitute for phosphoserine and phosphothre- onine has been observed previously with two proteins (25,40), although this approach has been successful in several other proteins (23-28). I t is possible that the spatial requirements are more stringent in some proteins and hence don't allow for the productive substitution of a phosphoamino acid with a negatively charged amino acid.

In the present studies the transiently expressed receptor mutant with tyrosine 1158 substituted with phenylalanine exhibited the same activation after autophosphorylation as the wild-type receptor (Fig. 5). This was true whether kinase activity was assessed with either poly(Glu,Tyr) or histone as exogenous substrate. Identical results were obtained when this mutant and the wild-type receptor were expressed in stable transfectants of NIH 3T3 cells and receptor kinase activation assessed after insulin treatment of intact cells (Fig. 6). Finally an independently produced mutant IR with tyrosine 1158 changed to aspartate also exhibited as much activation by autophosphorylation as the wild-type receptor (Fig. 5).

The autophosphorylation sites of the Y1lssF mutant IR were determined by two-dimensional tryptic phosphopeptide mapping (Fig. 7). The presence of bisphosphopeptides B2 and B3 encompassing tyrosines 1162 and 1163 shows that these 2 tyrosines can be rapidly autophosphorylated in the absence of tyrosine 1158. Thus this mutant receptor appears to be capable of achieving full kinase activation towards an exogenous substrate upon bisphosphorylation of the twin tyrosines 1162 and 1163.

Previously, it has been reported that full activation of the tyrosine kinase requires the trisphosphorylation of tyrosines 1158, 1162, and 1163 (15, 20). This apparent discrepancy could be most simply explained by assuming that tyrosine 1158 phosphorylates early in the cascade of phosphorylation of tyrosines 1158, 1162, and 1163. In support of this hypothesis, we have previously reported that only 10-15% of bisphosphopeptides from the wild-type human placental insulin receptor phosphorylated in vitro were phosphorylated on both tyrosines 1162 and 1163, the remainder being phosphorylated on tyrosine 1158 and either 1162 or 1163 (19). As a result, with either tyrosine 1162 or 1163 being phosphorylated last, full activation of the wild-type kinase would only occur upon trisphosphorylation of these 3 tyrosines.

A synthetic peptide encompassing tyrosines 1158,1162, and 1163 (RRDIYETDYYRK) is phosphorylated on all 3 tyrosines by a soluble derivative of the cytoplasmic tyrosine kinase domain of the human insulin r e ~ e p t o r . ~ Phosphorylation of this synthetic peptide is initiated at tyrosine 9 (equivalent of tyrosine 1162) and is followed by tyrosine 10 (1163) and finally by tyrosine 5 (1158). The apparent difference in the order of phosphorylation of the tyrosines in this peptide compared to that found in the intact wild-type IR, could be due to the fact that this sequence is held in a rigid conformation in the intact receptor p subunit with only tyrosines 1158 and either 1162 or 1163 initially accessible to the kinase active site rather than tumbling in solution as a synthetic peptide where all tyrosines are potentially accessible for phosphorylation.

The lack of a direct regulatory role for tyrosine 1158 in the activation of the receptor kinase is in apparent conflict with the recent studies of Wilden et al. (41). In the latter study a stable line of Chinese hamster ovary cells was generated which overexpressed the human IR with the comparable tyrosine 1158 to phenylalanine substitution. This mutant receptor was reported to have markedly reduced abilities to autophospho-

Levine, B. A., Clack, B., and Ellis, L., (1991) J. Bid. Chern., in press.

996 Tyrosines 1158, 1162, and 1163 and Insulin Receptor Kinase Activity

rylate and phosphorylate an exogenous substrate. Wilden et al. (41) used the human IR cDNA lacking exon 11 which codes for an additional 12 amino acids at the C terminus of the cy subunit in contrast to the cDNA used in our study which possesses this exon (42,43). Potentially, the conflict in the role of tyrosine 1158 could be explained by the presence of this exon in our IR. Since the two forms of the receptor are differentially expressed in various tissues and differ in their affinities for insulin (44), a difference in their regulation by autophosphorylation would have important physiological consequences.

In conclusion, the simplest interpretation of our results is that the phosphorylation of tyrosine 1158 does not have a direct productive effect on the activation of the IR tyrosine kinase. Rather, the role of tyrosine 1158 may be to closely regulate the rate of bisphosphorylation of the twin tyrosines 1162 and 1163 and thus full activation of the kinase. These 3 tyrosines are all present in a number of related tyrosine kinases including the Drosophila insulin receptor, the insulin- like growth factor-I receptor, seuenless, and u-ros (45) and, therefore, a similar role for these 3 tyrosines may not be unique to the insulin receptor.

Acknowledgments-We are grateful to Drs. K. Siddle for gifts of monoclonal antibody 83-7 and the polyclonal antibody to the carboxyl terminus of the insulin receptor (Ros-2), J. Pessin for the IR CMV cDNA, and Naoki Arai for the SRa vector.

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Documents

The Regulatory Role of Known Tyrosine Autophosphorylation Sites