Biochem. J. (1994) 298, 471-477 (Printed in Great Britain)

Identification of serines-967/968 in the juxtamembrane region of the insulinreceptor as insulin-stimulated phosphorylation sitesFeng LIU and Richard A. ROTH*Department of Pharmacology, Stanford University School of Medicine, Stanford, CA 94305, U.S.A.

A line of Chinese hamster ovary cells overexpressing proteinkinase Ca was transfected with cDNAs encoding either the wild-type human insulin receptor or one of two mutant insulinreceptors with either Ser-967 and -968 or -974 and -976 in thejuxtamembrane region changed to alanine. Both mutant recep-

tors exhibited normal insulin-activated tyrosine kinase activity as

assessed by either autophosphorylation or insulin-stimulatedincreases in anti-phosphotyrosine-precipitable phosphatidyl-inositol 3-kinase. The wild-type and mutant insulin receptorswere also examined for serine and threonine phosphorylationin response to insulin and activation of protein kinase C. Tovisualize Ser/Thr-phosphorylation sites of the receptor better inresponse to insulin, the receptor from in vivo-labelled insulin-treated cells was first treated with a tyrosine-specific phosphataseto remove all tyrosine phosphorylation. Phosphopeptides

INTRODUCTION

Insulin induces extensive tyrosine phosphorylation of the cyto-plasmic domain of the f-subunit of its receptor. At least sixtyrosines appear to be phosphorylated, including one in thejuxtamembrane region (Tyr-972), three in the kinase domain(Tyr-1158, -1162 and -1163) and two in the carboxy tail of thereceptor (Tyr- 1328 and - 1334) (Tavare and Denton, 1988; Avruchet al., 1990; Feener et al., 1993). The tyrosine phosphorylation ofresidues in the kinase domain appears to greatly activate theability of the receptor to phosphorylate other proteins, both invitro and in vivo, and to make the receptor kinase independent ofthe presence of insulin (Ellis et al., 1986; Avruch et al., 1990;Wilden et al., 1992). The tyrosine phosphorylation of residues inthe carboxy tail of the receptor appears to potentiate the abilityof the receptor to stimulate some responses but not others incertain cell types (Ando et al., 1992; Dickens et al., 1992; Takataet al., 1992). Phosphorylation of the juxtamembrane tyrosineappears not to affect receptor autophosphorylation but to affectthe ability of the receptor to mediate subsequent biologicalresponses and to phosphorylate various endogenous substratesincluding insulin receptor substrate 1 (IRS-1) (White et al., 1988;Feener et al., 1993).

In addition to this tyrosine phosphorylation, the insulinreceptor fl-subunit is also extensively phosphorylated on serineand threonine residues (Avruch et al., 1990; Pillay et al., 1991).The Ser/Thr phosphorylation of the receptor also occurs inresponse to insulin treatment of cells as well as when cells are

treated with activators of either protein kinase C (PKC) or cyclicAMP kinase (Jacobs and Cuatrecasas, 1986; Stadtmauer andRosen, 1986; Takayama et al., 1988). At least one threonine

from the three receptors were analysed by high-percentagepolyacrylamide/urea gel electrophoresis and two-dimensionalt.l.c. The mutant receptor lacking Ser-967 and -968 but not themutant lacking Ser-974 and -976 was found to be missingphosphorylated peptides in response to insulin and, to a lesserextent, after activation of protein kinase C. However, theinsulin-stimulated increase in anti-phosphotyrosine-precipitablephosphatidylinositol 3-kinase was inhibited to the same extent byactivation of protein kinase C in cells expressing the two mutantreceptors as in cells expressing the wild-type receptor. Theseresults indicate that these four serine residues in the juxta-membrane region are not major regulatory sites of the intrinsictyrosine kinase activity of the insulin receptor by protein kinaseC, although Ser-967 and/or -968 appear to be phosphorylated inresponse to insulin.

(Thr-1348) and two serines (Ser-1305/1306) in the carboxy tail ofthe receptor have been found to be phosphorylated in vitro bypurified PKC and in vivo after treatment of cells with eitherinsulin or activators of PKC (Koshio et al., 1989; Lewis et al.,1990a,b; Tavare et al., 1991; Ahn et al., 1993). The insulinreceptor has also been shown to be phosphorylated in vitro bycyclic AMP kinase (Roth and Beaudoin, 1987), a casein kinaseI-like enzyme (Rapuano and Rosen, 1991) and an insulin-stimulated Ser/Thr kinase that associates with the receptor buthas yet to be purified (Czech et al., 1988; Smith et al., 1988). Ithas also been reported that the insulin receptor itself may becapable of Ser/Thr autophosphorylation (Baltensperger et al.,1992). In contrast with the well-defined effects of tyrosinephosphorylation of the receptor on receptor functions, the effectsof Ser/Thr phosphorylation have been more controversial.Although some initial reports suggested that Ser/Thr phosphoryl-ation might affect insulin binding, most subsequent reportshave not confirmed this (Grunberger, 1991; Haring, 1991).Ser/Thr phosphorylation of the receptor has also been reportedin some studies to affect the receptor's intrinsic tyrosine kinaseactivity when assayed in vitro with artificial substrates (Takayamaet al., 1988; Karasik et al., 1990). In recent studies this could notbe confirmed, although it was found that the insulin-stimulatedincrease in anti-phosphotyrosine-precipitable phosphatidyl-inositol 3-kinase activity was decreased when the receptor was

extensively Ser/Thr phosphorylated by activation of PKC (Chinet al., 1992; Coghlan and Siddle, 1993). The inhibitory effect ofSer/Thr phosphorylation of the insulin receptor is of potentialimportance in the desensitization of the receptor, either inresponse to insulin itself or through the activation of othersignalling systems by counter regulatory hormones (Treadway

Abbreviations used: IRS-1, insulin receptor substrate-1; CHO, Chinese hamster ovary; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase

C; PTP ib, protein tyrosine phosphatase lb; GST, glutathione transferase.The amino acid numbering corresponds to that of Ebina et al. (1985).* To whom correspondence should be addressed.

471Biochem. J. (1994) 298, 471-477 (Printed in Great Britain)

472 F. Liu and R. A. Roth

et al., 1989; Issad et al., 1992). In addition, excessive Ser/Thrphosphorylation of the receptor, possibly as a result of activationof PKC, could even play a role in the decreased receptor kinaseactivity observed in many non-insulin-dependent diabeticpatients with insulin resistance (Caro et al., 1989; Thies et al.,1990; Haring, 1991; Inoguchi et al., 1992).A further delineation ofthe kinases responsible for this Ser/Thr

phosphorylation of the receptor, as well as the determination ofthe particular amino acid residues involved, could therefore beimportant in unravelling the pathogenesis of non-insulin-de-pendent diabetes. In the present study we report on the results ofthe mutagenesis of all four serine residues in the juxtamembraneregion of the insulin receptor. These serines were chosen because,as noted above, this region appears to play a critical role in therecognition of endogenous substrates such as IRS- 1. In addition,recent studies of Feener et al. (1993) indicated that serinephosphorylation occurred in this region of the receptor.The present studies indicate that Ser-967 and/or -968 are

phosphorylated in response to insulin and, to a lesser extent,on activation of PKC. However, phosphorylation of theseserines did not mediate the decrease in insulin-stimulatedanti-phosphotyrosine-precipitable phosphatidylinositol 3-kinaseobserved after activation of PKC (Chin et al., 1992).

MATERIALS AND METHODS

Site-directed mutagenesis and construction of expression vectors

The wild-type human insulin receptor cDNA (Ellis et al., 1986)was subcloned into pBluescript II SK plasmid (Stratagene) andused as the template to generate mutants S967/968A and S974A.Oligonucleotide-directed mutagenesis was carried out as de-scribed by Kunkel et al. (1987). Chemically synthesized oligo-nucleotides used for mutagenesis are as follows (the mismatchedbases are underlined): S967/968A, 5'-ACCGCTTTACGCT-GCAGCAAACCCTGAG-3' (new PstI site); S974A, 5'-CCTG-AGTATCTCGGTGCCAGTGA-3' (new Ban I site). To generatemutant S974/976A, a 2.4 kb BamHI-XbaI fragment was isolatedfrom the S974A mutant cDNA and subcloned into pBluescript II

SK plasmid. Double-stranded DNA site-directed mutagenesiswas carried out by using the Transformer Site-Directed Muta-genesis Kit (Clontech). The oligonucleotide used for muta-genesis was as follows (the mismatched bases are underlined):5'-CTGAGTATCTCGCTGCCGCTGATGTGTTTC-3'. Thetrans oligonucleotide used was: 5'-GGTCGACGGTCGCGAT-AAAGCTTGA-3' (to remove the ClaI site in the multiplecloning site of the pBluescript plasmid). The recombinant plas-mids containing the mutant receptor cDNAs were used totransform DH5L and the mutations and proper construction ofthe vectors were confirmed by DNA sequencing the mutatedregions. The mutant cDNAs were subcloned into the SR,expression vector (Takebe et al., 1988), and the recombinantplasmids were used for transfections.

Expression of PKCxc in Chinese hamster ovary (CHO) cell lines

Transfections were carried out as described previously (Chin etal., 1992). In brief, 5 x 105 CHO cells were grown in 100-mm-diam. plates and incubated overnight at 35 0C and 30% CO2.pECE-PKC. (a gift from Dr. P. Parker, ICRF Laboratory,London) (20 4ug) and 1,ug of pSV2neo were allowed to form a

calcium co-precipitate. The precipitate was added dropwise tothe cell medium and the cells were incubated for 4 h at 35 °C and3% CO2. After a glycerol shock, the cells were incubatedovernight at 37 °C and 5 % CO2. The cells were then split 1: 10

and placed in Ham's F-12 medium containing 10% (v/v)newborn-calf serum (Gibco), 100 ,ug/ml streptomycin and400,ug/ml G418 (Gibco). Positive colonies were identified by[3H]phorbol 12,13-dibutyrate binding and PKC activity asdescribed (Chin et al., 1992) and then cloned by limiting dilution.

Expression of the wild-type and mutant insulin receptors InCHO/PKC cell linesTransfections were carried out as described above. SRa insulinreceptor (20 ,tg) and 1 ,tg of PBSpacAp (de la Luna et al., 1988)were used in the transfections. Transfectants were selected with8 ,ug/ml puromycin. Positive colonies were identified by Westernblotting with anti-phosphotyrosine antibody RC-20 (SignalTransduction Laboratories) after insulin stimulation and con-firmed by Western blotting with anti-(insulin receptor) antibody.

Expression and purification of protein tyrosine phosphatase lb(PTP 1b)Plasmid pGEXKG, which encodes rat brain PTP lb fused toglutathione transferase (GST), was a gift from Dr. A. Saltiel(Warner-Lambert Co., Ann Arbor, MI, U.S.A.). For expressionand purification of the GST-PTP lb fusion protein, the plasmidwas used to transform Escherichia coli strain DH5,. Overnightcell cultures (100 ml) containing the plasmid were diluted 1:10and grown at 28 °C for 1 h and 20 min. Expression ofGST-PTPlb fusion protein was induced by addition of isopropyl /J-D-thiogalactoside to a final concentration of 1 mM. After a further3.5 h, the cell culture was harvested, washed with 10 mMTris/HCl, pH 8.0, and the cell pellet was resuspended in lysisbuffer containing 50 mM Tris/HCl, pH 7.5, 1 mM dithiothreitol,5 mM EDTA, 50 mM KCI, 1 mM phenylmethanesulphonylfluoride, 1% (v/v) Triton X-100 and 1 mg/ml lysozyme. After30 min at 0 °C, the lysate was sonicated for 5 min at 0 'C. Thecell debris was removed by centrifugation at 12000 g for 15 min.The GST-PTP lb fusion protein was purified by incubating thesupernatant with 1 ml of glutathione-agarose (Pharmacia) over-night at 4 'C. After the agarose beads had been washed threetimes with PBS (5 mM Na2HPO4, pH 7.4, 15 mM NaCl), theGST-PTP lb fusion protein was eluted with 50 mM Tris/HCl,pH 8.0, containing 10 mM GSH. The purified enzyme was storedat -20 'C in 20% (v/v) glycerol.

Radiolabelling and PTP lb treatment of 32P-labelled insulinreceptor in vivoMonolayers of cells were grown in 100-mm-diam. plates toconfluence. After incubation in phosphate-free Krebs-Ringerbicarbonate buffer (KRBB; 20 mM Hepes, pH 7.6, 3 mM CaCl2,5 mM KCl, 7 mM NaHCO3, 107 mM NaCl, 1 mM MgSO4,10 mM glucose, 0.1 0% BSA) for 30 min at 37 'C, the cells wereradiolabelled with 0.3 mCi of carrier-free [32P]orthophosphate/plate for 3 h at 37 'C. Insulin (10 nM) was added during the last8 min of radiolabelling. The cells were then placed on ice, washedtwice with ice-cold KRBB, and lysed with 0.6 ml/plate lysisbuffer containing 50 mM Hepes, pH 7.6, 150 mM NaCl, 1%Triton X-100, 10 mM NaF, 50 mM ,-glycerophosphate, 1 mg/ml bacitracin, 1 mM phenylmethanesulphonyl fluoride and 1 mMN-ethylmaleimide. Cell debris was pelleted at 14000 g for 10 minand the supernatant fraction was immunoprecipitated overnightat 4 'C with 5 ,jg each of the anti-(insulin receptor) monoclonalantibodies 5D9 and 29B4 bound to Protein G-agarose. Theimmunoprecipitates were washed twice with buffer A (20 mMNa2HP04, pH 8.6, 0.5% Triton X-100, 0.1% SDS, 0.02%

Serine-phosphorylation sites in the insulin receptor

kDa

97 -

68 -

43

25

(a)A B C D

- 4 k *

_

(b)A B C D

I I

(a)A B C D

I....

...

4;

(b)Peptide A B C

-4i

-ii

-5i-6i

-2i

-3i

Figure 1 Expression of the wild-type and mutant insulin receptors

(a) Immunoblotting of the insulin receptors. Lysates of insulin-treated parental CHO cells (laneA), cells overexpressing the wild-type receptor (lane B) or cells overexpressing mutantS967/9768A (lane C) or mutant S974/976A (lane D) were immunoprecipitated with monoclonalanti-receptor antibodies and the precipitates were electrophoresed on 10% (w/v) polyacrylamide/SDS gels, transferred to nitrocellulose and immunoblotted with a monoclonal antibody to thefl-subunit of the insulin receptor. (b) Anti-phosphotyrosine immunoblotting. The same blot as

in (a) except that it was probed with anti-phosphotyrosine antibody RC-20.

(a)

.kDa97

68 -

43 -

(b)

Insulin - - + +

PTP 1b - + - +

kDa-97

-68

-43

-25

Insulin - - + +

PTP lb - + - +

Figure 3 Analysis of the phosphorylated tryptic peptides of the wild-typeand mutant Insulin receptors by high-percentage polyacrylamide/urea gelelectrophoresis

In vivolabelled cells were treated with either 10 nM insulin (a, lanes B-D) or 1.6 /WM PMA(a, lane A; b, lanes A-C). The insulin receptors from cells overexpressing the wild-type receptor(a, lanes A and B; b, lane A), mutant S967/968A (a, lane C; b, lane B) or mutant S974/976A(a, lane D; b, lane C) were immunoprecipitated, treated with the PTP lb and cleaved withtrypsin as described in the Materials and methods section. The resulting tryptic peptides were

analysed by high-percentage polyacrylamide/urea gel electrophoresis.

(c)

PTP 1b +

- P-Ser SOS/PAGE and Western-Blot analysis-P-Thr SDS/PAGE was performed by the procedure of Laemmli (1970)

using 10% (w/v) polyacrylamide. After electrophoresis, proteinswere electrophoretically transferred to nitrocellulose membranes

- P-Tyr for Western blotting or Immobilon P for phosphoamino acid

analysis. The immunoblots were blocked for 1 h at room

temperature in a solution containing 10 mM Tris/HCl, pH 7.5,154 mM NaCl, 3% BSA and 0.1 % Tween 20 and then incubatedovernight at 4 °C with monoclonal anti-receptor antibody 2H2directed against the f-subunit of the insulin receptor (a gift fromDr. K. Shii, Hyogo Institute, Aitashi, Japan). After incubationwith horseradish peroxidase-conjugated anti-(mouse IgG) anti-body for 1 h at room temperature, the membrane was developedwith the ECL kit (Amersham).

Figure 2 Treatment of receptor with PTP lb

(a) Analysis of in vivo]abelled receptor. Metabolically labelled cells overexpressing the wild-type insulin receptor were treated wtih 10 nM insulin for 8 min as indicated. The insulinreceptor from the cells was immunoprecipitated and treated with PTP lb as indicated. Theprecipitates were electrophoresed on a 10% (w/v) polyacrylamide/SDS gel and transferred toImmobilon P; its autioradiogram is shown. (b) Analyses of PTP lb-treated receptor byimmunoblotting. The same blot as shown in (a) was immunoblotted with the anti-phosphotyrosineantibody RC-20. (c) Phosphoamino acid analysis of the PTP 1 b-treated receptor. In vivo]abelledreceptor from the insulin-treated cells was treated with or without PTP 1 b, hydrolysed and thephosphoamino acids were determined.

NaN3) containing 1 M NaCl and 0.1 0% BSA and then twice withbuffer A containing 0.15 M NaCl.For PTP lb treatment, the immunoprecipitates were resus-

pended in a buffer containing 25 mM imidazole/HCl, pH 7.0,10 mM dithiothreitol, 0.15 M NaCl, 1 mg/ml BSA and 0.5 ,uMokadaic acid (Calbiochem). The reaction mixture was incubatedat 30 °C for 15 min. Optimal conditions for removal of all thephosphotyrosine were previously determined by varying theamounts of enzyme and the time of incubation.

Phosphoamino acid analysisThe insulin receptor on the Immobilon P membrane was hydro-lysed under vacuum for 2.5 h at 110 °C in 1 ml of 6 M HCl(Pierce Chemical Co.). The membrane was rehydrated by theaddition of 1 drop of methanol, and the amino acids were elutedby incubating the membrane with 1 ml of water for 1 h at roomtemperature or overnight at 4 'C. After lyophilization, the samplewas resuspended in 5,ul of phosphoamino acid standards con-

taining phosphotyrosine, phosphoserine and phosphothreonineand spotted on to t.l.c. plates. Thin-layer electrophoresis was

carried out at 1500 V for 55 min in acetic acid/pyridine/water(10:1:189, by vol.). The plates were dried and exposed to X-rayfilms.

Tryptic digestion, high-percentage PAGE and two-dimensionalphosphopeptide mappingTryptic digestion of 32P-labelled human insulin receptor was

conducted as described by Luo et al. (1991). In brief, the

473

Peptide

-4t-it- 5t- 6t

-2t-3t

**.*No, 1-*-/.-Subunitso.

-1

.0

474 F. Liu and R. A. Roth

{a})==

4BR,.

N, ..C:o.%

+ - Electrophoresis

Figure 4 Two-dimensional mapping of the phosphorylated tryptic peptides of the wild-type (a) and mutant S967/968A (b) Insulin receptors after Insulintreatment

Immunoprecipitated receptor from metabolically labelled cells was treated with PTP lb, cleaved with trypsin and analysed as described in the Materials and methods section. A schematic diagramof the observed peptides is shown in (c). Peptide T is so called as it migrates in a position that corresponds to that of a peptide containing phosphothreonine although this peptide has not beenshown to contain phosphothreonine in the present work.

radiophosphorylated insulin receptor was subjected to SDS/PAGE, blotted on to nitrocellulose membranes and digestedwith 20 ,ug of TosPheCH2Cl ('TPCK')-treated trypsin (Worth-ington) in a freshly prepared 50 mM NH4HCO3 buffer at 37 °Cfor 18 h. An additional g of TosPheCH2Cl-treated trypsinwas added and the reaction mixture was incubated for a further6 h. The tryptic digests were lyophilized, resuspended in a gelloading buffer containing 0.125 M Tris/HCl, pH 6.8, 6 M urea

and Bromophenol Blue. High-percentage alkaline polyacryl-amide gels were prepared as described by West et al. (1984).Electrophoresis was carried out overnight at a constant currentof 1O mA. The gel was dried under vacuum and autoradio-graphed. For two-dimensional tryptic phosphopeptide map-

ping, the lyophilized sample was resuspended in 2-3 ,ul of watercontaining 5 mg/ml dinitrophenyl-lysine and 1 mg/ml xylenecyanol FF and spotted on a 100 ,um 20 cm x 20 cm cellulose t.l.c.plate (E. Merck, catalogue no. 5716). First-dimension separationwas carried out at pH 3.5 in acetic acid/pyridine/water(10:1:189, by vol.) (Boyle et al., 1991). The electrophoresis was

conducted at 400 V for 2 h. The second-dimension t.l.c. was

carried out for 8-10 h in a buffer containing butan-l-ol/pyridine/acetic acid/water (15:10:3:12, by vol.). The plateswere dried and autoradiographed.

RESULTS

For the present studies, a line ofCHO cells overexpressing PKCawas developed. By phorbol binding, these cells expressed approx.seven times as much PKC as the parental CHO cells. This cellline was then transfected with cDNAs encoding the wild-typehuman insulin receptor, a mutant insulin receptor with Ser-967and -968 changed to alanine (S967/968A) or a mutant insulinreceptor with Ser-974 and -976 changed to alanine (S974/976A).Cell lines overexpressing the three different receptors were thencharacterized. By Western blotting with an antibody to the f-

subunit (Figure la), all three cell lines were found to express highlevels of insulin receptor. The cells expressing the wild-typereceptor and mutant S974/976A were found to contain approx.the same amount of receptor, whereas mutant S967/968Aexpressed approx. two to three times less receptor (Figure la).Both mutant receptors appeared to be processed normally, as thefraction of proreceptor and the sizes of their f-subunits were

normal (Figure Ia). The receptor tyrosine kinase activities of thetwo mutant receptors also appeared to be unaffected, as insulinstimulated comparable increases in the extent of tyrosine auto-phosphorylation in the three receptors (Figure lb).To assess the insulin-stimulated serine phosphorylation of the

(b)I

(c)

CLc06-o

a0

E0

u

...........

Serine-phosphorylation sites in the insulin receptor 475

-CaC-

0

E0L.-

+ 4 Electrophoresis f-

Figure 5 Two-dimensional mapping of the phosphorylated tryptic peptides of the wild-type (a) and mutant S967/968A (b) Insulin receptors after activatonof PKC

Immunoprecipitated receptors from metabolically labelled cells treated with 1.6 /,cM PMA were cleaved with trypsin and analysed as described in the Materials and methods section. A schematic

diagram of the observed peptides is shown in (c).

receptors in vivo, a method was sought that would allow a betterexamination of the serine-phosphorylation sites without thecomplication of insulin-stimulated tyrosine phosphorylation. Tothis end, receptor from insulin-treated cells was immunoprecipi-tated and then treated with a recombinantly produced tyrosine-specific phosphatase in the presence of an inhibitor of serinephosphatases, okadaic acid (Cohen et al., 1990). This procedurewas found to remove all of the detectable tyrosine phosphoryl-ation without affecting the extent of serine phosphorylation ofthe receptor (Figure 2). For in vivo-labelled cells, approx. 60% ofthe counts were removed from the receptor from insulin-treatedcells and no decrease was observed in the receptor from phorbol12-myristate 13-acetate (PMA)-stimulated cells.The wild-type and two mutant receptors from in vivo-labelled

insulin-treated cells were isolated by immunoprecipitation,treated with the tyrosine-specific phosphatase and digested withtrypsin. The labelled peptides were analysed by high-percentagepolyacrylamide/urea gel electrophoresis. Wild-type receptorfrom insulin-treated cells exhibited three major (peptides li-3i)and three minor (peptides 4i-6i) bands (Figure 3a). One major(2i) and two minor (4i and 6i) bands were missing from mutantS967/968A. Mutant S974/976A had the same phosphopeptidebands as the wild-type receptor (Figure 3a). Wild-type receptorfrom PMA-stimulated cells exhibited two major bands (peptidesIt and 3t) with the same mobility as two bands (peptides Ii and3i) from the wild-type receptor of insulin-treated cells (Figure 3).Mutant receptor S974/976A from PMA-stimulated cellsexhibited the same pattern as the wild-type receptor, whereasmutant S967/968A lacked one of the minor phosphopeptidebands (peptide 4t) present in the wild-type receptor (Figure 3b).To confirm the differences in the pattern of phosphopeptide

tryptic bands observed in polyacrylamide/urea gel electro-

phoresis between the wild-type and mutant S967/968A receptors,tryptic peptides from these two receptors were also analysed bytwo-dimensional t.l.c. By this technique, mutant S967/968A was

also observed to lack one major phosphopeptide (peptide b) ininsulin-treated cells (Figure 4) and two minor spots in PMA-treated cells (Figure 5).The two mutant receptors were also tested for their ability to

mediate the insulin-stimulated increase in anti-phosphotyrosine-precipitable phosphatidylinositol 3-kinase activity. The mutantreceptors were found to behave like the wild-type receptor in thisassay (Figure 6). Although the serines in the juxtamembraneregion did not appear to be major sites of PMA-stimulatedphosphorylation (Figures 3 and 5), we also verified that themutant receptors were regulated in the same way as the wild-typereceptor after activation of PKC. To this end, the three cell lineswere treated with various concentrations ofinsulin in the presenceor absence of PMA, lysed and the lysates were adsorbed withanti-phosphotyrosine antibodies. The resulting precipitates were

assayed for phosphatidylinositol 3-kinase activity. PMA treat-ment caused an approx. 60-70% inhibition of the insulin-stimulated increase in this activity in the cells expressing the wild-type receptor as well as the cells expressing the two mutantreceptors (Figure 6).

DISCUSSIONAlthough extensive studies have documented the existence of atleast six tyrosine autophosphorylation sites in the insulin receptor(Avruch et al., 1990), much less success has been reported on theidentification of the many serine- and threonine-phosphorylationsites in the receptor. The possibility that Ser/Thr phosphorylationof the receptor regulates its intrinsic tyrosine kinase activity and

(a) (b) I.~~~~~~~~~~ 4

.. :

.}~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.

. .--p

(c)ODinitrophenyl-lysi ne

Xylene cyanol FF0 a

(: b'

<=>d' s1eI

0Orhi° Origin

476 F. Liu and R. A. Roth

120

100 F

80

60 r

40 F

20

0

:3120Ex

1000

ol

o-i*, 80._..

0 60

o 40

0

> 20

cn

0' 6

120

100

80

60

40

20

[insulin] (nM)

Figure 6 PMA-mediated InhibItIon of the insulin-stimulated increasein ant-phosphotyrosine-precipitable phosphatldylinositol 3-kinase Incells expressing (a) wild-type, (b) mutant 3967/968A or (c) mutantS974/976A Insulin receptors

The indicated cells were treated with the various concentrations of insulin in the presence (-)or absence (0) of 1.6 1sM PMA. For each cell type, the results shown have been normalizedto the amount of phosphatidylinositol 3-kinase activity present in the precipitates from cellstreated with 10 nM insulin in the absence of PMA.

subsequent signal-transducing abilities after either heterologousand/or homologous desensitization and possibly in type-IIdiabetes makes the identification of these sites an important goal.

In the present work we report on our attempts to identify serine-phosphorylation sites in the juxtamembrane region of the re-

ceptor. Previous studies have identified threonine (Thr- 1348) andserine (Ser-1305/1306) phosphorylation sites in the carboxy tailof the receptor (Koshio et al., 1989; Lewis et al., 1990a,b; Tavareet al., 1991; Ahn et al., 1993). However, recent studies haveindicated that the juxtamembrane region plays a critical role indetermining the ability of the receptor to phosphorylate anendogenous substrate (IRS-1) and mediate biological signallingand that both tyrosine- and serine-phosphorylation sites are

present in this region of the receptor (White et al., 1988; Feeneret al., 1993). Thus phosphorylation of one of the four serines(Ser-967, -968, -974 and -976) in this region could affect receptorsignalling.

In the present study, we report on the characterization of twomutant receptors in which the four serines have been mutated. Inone mutant, Ser-967 and -968 have been changed to alanine, andin the other mutant, Ser-974 and -976 have been mutated. Bothmutant receptors appear to be expressed and processed normally(Figure 1). In addition, both mutant receptors appear to auto-phosphorylate normally and to mediate the phosphorylation ofan endogenous substrate as assessed by the insulin-stimulatedincrease in anti-phosphotyrosine-precipitable phosphatidyl-inositol 3-kinase activity (Figures 1 and 6). These results suggestthat the structure of these two mutant receptors has not beendrastically altered by mutagenesis.The identification ofinsulin-stimulated serine-phosphorylation

sites in the insulin receptor is complicated by the extensivetyrosine phosphorylation that accompanies insulin treatment. Inthe present studies we show that the tyrosine phosphorylationcan be mostly eliminated without affecting the serine phosphoryl-ation of the receptor by the use of a recombinantly producedtyrosine phosphatase (Figure 2). By utilizing this procedure, wecan show that insulin treatment of cells results in the phosphoryl-ation of multiple serine sites in the receptor (Figure 3). A majorfraction (approx. 20 %) of the serine phosphorylation is lost inthe mutant receptor lacking Ser-967/968. This correlates withthe loss of one major site by two-dimensional t.l.c. (Figure 4)and one major and two minor bands on high-percentagepolyacrylamide/urea gel electrophoresis (Figure 3). The presenceof three bands on this gel that are affected by the mutagenesiscould be due to heterogeneity in the charge of this peptideand/or its partial cleavage, as these gels separate by a mixture ofcharge and size. In regard to this latter possibility, it should benoted that both the arginine preceding and the one following thejuxtamembrane tryptic peptide are poor sites for trypsin cleavage,as they are either in a cluster with other basic residues orfollowed by a glutamic acid respectively (Boyle et al., 1991). Theinability to detect the two minor peptides by two-dimensionalt.l.c. could be due to these peptides being poorly resolved in thissystem. In summary, we have found by two different proceduresthat a mutant receptor lacking Ser-967 and -968 exhibits a loss inphosphopeptide(s) observed after insulin stimulation. The sim-plest explanation for these results is that Ser-967 and/or -968 are

phosphorylated in response to insulin treatment. In contrast,mutant S974/976A did not show a loss of any of the majorserine-phosphorylated bands (Figure 3), indicating that theseserines are not major sites for insulin-stimulated phosphoryl-ation.

Activation of PKC in cells has also been shown to result in anincrease in the Ser/Thr phosphorylation of the receptor (Jacobsand Cuatrecasas, 1986; Takayama et al., 1988; Pillay et al.,1991). To ensure a high level of Ser/Thr phosphorylation of thereceptor by this kinase, we have utilized a line of CHO cells

which overexpresses PKCa to express the mutant and wild-type

(a)

wo-PMA a

li--

-4

+PMA

0 10

2 a

1010

Serine-phosphorylation sites in the insulin receptor 477

insulin receptors. Previous studies have shown that cells over-expressing this enzyme exhibit 3- to 4-fold higher levels ofSer/Thr phosphorylation of the receptor in response to activatorsof PKC (Chin et al., 1992). Even with this high level of Ser/Thrphosphorylation, the mutant S967/968A only exhibited a loss ofa minor phosphorylated peptide in PMA-treated cells (Figure 3).These results indicate that these serines are not major phosphoryl-ation sites in cells with activated PKC. Mutants S974/976A didnot exhibit any difference in phosphorylation in response toPMA (Figure 3), indicating that these two serines are notphosphorylated in response to activation of PKC.Although the serines in the juxtamembrane region did not

appear to be major phosphorylation sites in response to activationof PKC, it is still possible that they are important regulatoryphosphorylation sites. Although some reports have indicatedthat a decrease in receptor kinase activity could be observed invitro after isolation of the receptor from cells with activated PKC(Takayama et al., 1988; Karasik et al., 1990), more recent studiesindicate that a better monitor of the inhibitory effect of excessiveSer/Thr phosphorylation of the receptor is a decrease in theability of the receptor to mediate the tyrosine phosphorylation ofan endogenous substrate. This can be most readily monitored bymeasuring the amount of phosphatidylinositol 3-kinase in anti-phosphotyrosine precipitates from insulin-treated cells (Chin etal., 1992). In agreement with these previous studies, a decrease ininsulin-stimulated anti-phosphotyrosine-precipitable phospha-tidylinositol 3-kinase activity was observed after PMA treatmentof cells overexpressing the wild-type insulin receptor and PKCa(Figure 6). The extent of inhibition of this response was approxi-mately the same in the cells expressing the two mutant receptors(60-70%) as was observed in the cells expressing the wild-typereceptor (Figure 6). These results are consistent with the abovedescribed findings, indicating that the serines in the juxta-membrane region are not phosphorylated to a great extent afteractivation of PKC. Additional studies are therefore required toidentify the other serine-phosphorylation sites of the receptorinduced by activation of PKC and to determine which, if any, ofthese are important in regulating the intrinsic tyrosine kinaseactivity of the receptor.

We thank Dr. Peter Parker for the cDNA encoding PKCx, Dr. Kozui Shii for themonoclonal anti-receptor antibody 2H2, Dr. John Glenney for a gift of Py2O and Dr.Alan Saltiel for the cDNA encoding the tyrosine phosphatase. This work wassupported by NIH grant 34926.

REFERENCESAhn, J., Donner, D. B. and Rosen, 0. M. (1993) J. Biol. Chem. 268, 7571-7576Ando, A., Momomura, K., Tobe, K., Yamamoto-Honda, R., Sakura, H., Tamori, Y., Kaburagi,

Y., Koshio, O., Akanuma, Y., Yazaki, Y., Kasuga, M. and Kadowaki, T. (1992) J. Biol.Chem. 267, 12788-12796

Received 2 August 1993/27 September 1993; accepted 6 October 1993

Avruch, J., Tornqvist, H. E., Gunsalus, J. R., Yorkow, E. J., Kyriakis, J. M. and Price, D. J.(1990) Handbook of Experimental Pharmacology: Insulin, pp. 313-366, Springer-Verlag,Berlin and Heidelberg

Baltensperger, K., Lewis, R. E., Woon, C.-W., Vissavajjhala, P., Ross, A. H. and Czech,M. P. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7885-7889

Boyle, W. J., van der Geer, P. and Hunter, T. (1991) Methods Enzymol. 201, 110-149Caro, J. F., Dohm, L. G., Pories, W. J. and Sinha, M. K. (1989) Diabetes/Metab. Rev. 5,

665-689Chin, J. E., Dickens, M., Tavare, J. M. and Roth, R. A. (1992) J. Biol. Chem. 268,

6338-6347Coghlan, M. P. and Siddle, K. (1993) Biochem. Biophys. Res. Commun. 193, 371-377Cohen, P., Holmes, C. F. and Tsukitani, Y. (1990) Trends Biochem. Sci. 5, 98-102Czech, M. P., Klarlund, J. K., Yagaloff, K. A., Bradford, A. P. and Lewis, R. E. (1988)

J. Biol. Chem. 263, 11017-11020de la Luna, S., Soria, I., Pulido, D., Oain, J. and Jimenez, A. (1988) FEBS Lett. 62,

121-126Dickens, M., Chin, J. E., Roth, R. A., Ellis, L., Denton, R. M. and Tavare, J. M. (1992)

Biochem. J. 287, 201-209Ebina, Y., Ellis, L., Jarnagin, K., Edery, M., Graf, L., Clauser, E., Ou, J., Masiarz, F., Kan,

Y. W., Goldfine, I. D., Roth, R. A. and Rutter, W. J. (1985) Cell 40, 747-758Ellis, L., Clauser, E., Morgan, D. O., Edery, M., Roth, R. A. and Rutter, W. J. (1986) Cell

45, 721-732Feener, E. P, Backer, J. M., King, G. L., Wilden, P. A., Sun, X. J., Kahn, C. R. and White,

M. F. (1993) J. Biol. Chem. 268, 11256-11264Grunberger, G. (1991) Cell Signalling 3,171-177Haring, H. U. (1991) Diabetologia 34, 848-861Inoguchi, T., Battan, R., Handler, E., Sportsman, R., Heath, W. and King, G. L. (1992) Proc.

Natl. Acad. Sci. U.S.A. 89, 11059-11063Issad, T., Young, S. W., Tavare, J. M. and Denton, R. M. (1992) FEBS Lett. 296, 41-45Jacobs, S. and Cuatrecasas, P. (1986) J. Biol. Chem. 261, 934-939Karasik, A., Rothenberg, P. L., Yamada, K., White, M. F. and Kahn, C. R. (1990) J. Biol.

Chem. 265, 10226-10231Koshio, I., Akanuma, Y. and Kasuga, M. (1989) FEBS Left. 254, 22-24Kunkel, T. A., Roberts, J. D. and Zakour, R. A. (1987) Methods Enzymol. 154, 367-382Laemmli, U. K. (1970) Nature (London) 227, 680-685Lewis, R. E., Caro, L., Perregaux, D. and Czech, M. P. (1990a) Biochemistry 29,

1807-1813Lewis, R. E., Wu, G. P., MacDonald, R. G. and Czech, M. P. (1990b) J. Biol. Chem. 265,

947-954Luo, K., Hurley, T. R. and Sefton, B. M. (1991) Methods Enzymol. 201, 149-152Pillay, T. S., Whittaker, J., Lammers, R., Ullrich, A. and Siddle, K. (1991) FEBS Lett. 288,

206-211Rapuano, M. and Rosen, 0. M. (1991) J. Biol. Chem. 266, 12902-12907Roth, R. A. and Beaudoin, J. (1987) Diabetes 36, 123-126Smith, D. M., King, M. J. and Sale, G. J. (1988) Biochem. J. 250, 509-519Stadtmauer, L. and Rosen, 0. M. (1986) J. Biol. Chem. 261, 3402-3407Takata, Y., Webster, N. J. and Olefsky, J. M. (1992) J. Biol. Chem. 267, 9065-9070Takayama, S., White, M. F. and Kahn, C. R. (1988) J. Biol. Chem. 263, 3440-3447Takebe, Y., Seiki, M., Fujisawa, J., Hoy, P., Yokoto, T., Arai, K., Yoshida, M. and Arai, N.

(1988) Mol. Cell. Biol. 8, 466-472Tavare, J. M. and Denton, R. M. (1988) Biochem. J. 252, 607-615Tavare, J. M., Zhang, B., Ellis, L. and Roth, R. A. (1991) J. Biol. Chem. 266,

21 804-21 809Thies, R. S., Molina, J. M., Ciaraldi, T. P., Freidenberg, G. R. and Olefsky, J. M. (1990)

Diabetes 39, 250-259Treadway, J. L, Whittaker, J. and Pessin, J. E. (1989) J. Biol. Chem. 264, 15136-15143West, M. H. P., Wu, R. S. and Bonner, W. M. (1984) Electrophoresis 5, 133-138White, M. F., Livingston, J. N., Backer, J. M., Lauris, V., Dull, T. J., Ullrich, A. and Kahn,

C. R. (1988) Cell 54, 641-649Wilden, P. A., Siddle, K., Haring, E., Backer, J. M., White, M. F. and Kahn, C. R. (1992)

J. Biol. Chem. 267, 1371 9-1 3727