Richard M. LevensonSBlf and Perry J. Blackshear$]]**. From the $Howard Hughes Medical Institute Laboratories, Durham, North Carolina 27710, the §Department of ...... Perrotti, N., Accili, D., Marcus Samuels, B., Rees Jones, R. W.,.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry andMolecular Biology, Inc.
Vol. 264, No. 33, Issue of November 25, pp. 19984-19993,1989 Printed in U.S.A.
Insulin-stimulated Protein Tyrosine Phosphorylation in Intact Cells Evaluated by Giant Two-dimensionalGel Electrophoresis* (Received for publication, June 7, 1989)
Richard M. LevensonSBlfand Perry J. Blackshear$]]** From the $Howard Hughes Medical Institute Laboratories, Durham, North Carolina 27710, the §Department of Pathology and the (ISectionof Diabetes and Metabolism, Divisionof Endocrinology, Metabolism,and Genetics, Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
We have studiedthe insulin-stimulated phosphoryla- that possesses intrinsic tyrosine kinase activity. Almost imtion of proteins inNIH 3T3 cells expressing high num- mediately after binding insulin, the receptor undergoes autobers of human insulin receptors (HIR 3.5 cells) using phosphorylation on tyrosine residues (3-6). Autophosphorythe technique of giant two-dimensional gel electropho- lation leads to increased protein tyrosine kinase activity of resis. In serum-deprived cells, insulin stimulated the the receptor toward exogenous substrates (7, 8), and this phosphorylation of more than 25 proteins; all but two increased kinase activity appears to be required for the genof these were also phosphorylated in response to 15% eration of intracellular signals in response to insulin. Many (v/v) fetal bovine serum, which also stimulated the investigators have shown that receptors mutated in regions phosphorylation of additional proteins thought to be C. In cells pre- required for kinase activity fail to transduce metabolic redirect substrates for protein kinase sponses to insulin when transfected into cells (9-13). The treated with the phosphatase inhibitor phenylarsine oxide, insulin specifically stimulatedthe phosphoryla- signaling events that follow binding of insulin to its receptor tion of at least 26 predominantly cytosolic proteins, and stimulation of its kinase activity, however, remain obonly oneof which was observed in insulin-treated cells scure. not exposed to phenylarsineoxide. Serum was without The search for the postulated direct intracellular phosphotyrosine substrates for the activated insulin receptor kinase effect in cells pretreated with phenylarsineoxide. Inphenylarsineoxide-pretreated cells, phosphoa- has been intense; although a numberof candidates have been mino acid analysis of 10 of the most highly labeled identified, the evidence linking thesewith metabolic pathways insulin-stimulated phosphoproteins showed that all10 stimulated by insulin is to dateonly circumstantial. Examples were labeled predominantlyor exclusively on tyrosine of such putative substrates include: 1)a protein of M,185,000 residues. The phosphorylation of several of these could in Fao hepatoma cells (14,15) and proteins of similar apparent be stimulated in vitro by the addition of insulin to a molecular weight in human epidermoid carcinoma cells (16), detergent extractof cells in the presence of Mn2+and 3T3-Ll adipocytes (17), and mouse neuroblastoma cells (18); ATP. In general, the insulin-stimulated phosphoryla- 2) a 46-kDa membrane protein in intact fat cells (19); and 3) tions observed in the presence of phenylarsine oxide were more rapid than those observed in its absence. a 120-kDa glycoprotein (pp120) in hepatoma cells (20). Other of other growth factors and mitogens insulin-stimulated phosphoproteins have recently been idenFinally, a variety did not stimulate any of the insulin-stimulated phos- tified by high affinity anti-phosphotyrosine antibodies, and phorylations in the presence of phenylarsine oxide. theseproteins are presumably phosphorylated ontyrosine Thus, the use of this inhibitor apparently unmaskeda (21, 22). An additional putative substrate for the insulin receptor number of novel insulin-specific protein phosphorylations that were ordinarily undetectable. We suggest has been described (23) in insulin-treated 3T3-Lladipocytes that atleast some of these proteinsmay be direct sub- pretreated with phenylarsine oxide (PAO),’ a cell-permeable strates for the insulin receptor protein tyrosine kinase trivalent arsenical compound that complexes with vicinal and may play significant roles in insulin action. dithiols including sulfhydryl groups on proteins (24). P A 0 was shown to inhibit insulin-stimulated hexose uptake in 3T3-Ll adipocytes (25) and to inhibit the insulin-stimulated Insulin is a polypeptide hormone that induces a wide variety serine phosphorylation of two proteins (26). In these experiof responses in target tissues including stimulation of amino ments, theauthors also found a novel insulin-stimulated acid and hexose uptake, phosphorylation and dephosphoryl- phosphorylation of a 15-kDa protein. This protein was phoscourse ation of enzymes involved in metabolic pathways, and in- phorylated on tyrosine residues with arapidtime creased synthesis of cellular constituents including lipids, intermediate between the earliest detectable event ( i e . autoproteins, RNA, and DNA (1, 2). As a first step in initiating phosphorylation of the insulin receptor) and onset of inthese responses, insulin binds to a specific cell surface receptor creased hexose uptake, which occurred about 3 min after the addition of insulin. Pulse-chase experiments indicated that * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ll Senior Associate of the Howard Hughes Medical Institute. ** Investigator of the Howard Hughes Medical Institute. To whom all correspondence should be addressed Box 3897, Duke University Medical Center, Durham, NC27710. Tel.: 919-684-8760; Fax: 919684-5458.
The abbreviations are: PAO, phenylarsine oxide; DMEM, Dulbecco’smodifiedEagle’s medium; EF-2, elongation factor 2; EGF, epidermal growth factor; EGTA, [ethylenebis(oxyethylenenitrilo)] tetraacetic acid FGF, fibroblast growth factor; HEPES, N-2-hydroxyethylpiperazine-N”2-ethanesulfonic acid; IGF-1, insulin-like growth factor 1; IR, insulin receptor; PDGF, platelet-derived growth factor; PIP, phenylarsine oxide-insulin-phosphoprotein; SDS, sodium dodecyl sulfate.
19984
Phosphorylation Insulin-stimulated Tyrosine
19985
Subcellular Fractionation-HIR 3.5 cells were grown to confluence in two 100-mm dishes, labeled with [32P]orthophosphate, and pretreated with P A 0 for 10 min and then with or without insulin for an additional 15 min. At the end of this incubation, the medium was aspirated, and the cells were quickly washed three times in 4 ml of ice-cold phosphate-buffered saline containing P A 0 (35 p M ) and then twice in swelling buffer (10 mM Tris/HCI (pH 7.9), 1.5 mM MgClz, 10 mM KC1, 35 PM PAO). The cells were swollen in 0.8 ml of swelling buffer on ice for 2 min and thenharvested with a rubber“policeman.” The cells were disrupted by 10 passes through a26-gauge needle and then transferred tomicrocentrifuge tubes containing EDTA (togive a final concentration of 10 mM) and sucrose (to give a final concentration of 0.1 M). Unbroken cells and nuclei were collected by centrifugation at 3,000 X g for 2 min at 4 “C. After removing the supernatants, the pellets were treated with 15% (w/v) trichloroacetic acid for 30 min, centrifuged at 3,000 X g for 1 min, and dissolved in two-dimensional gel lysis buffer (“crude nuclei”). The supernatants were then centrifuged at 12,000 X g for 20 min at 4 “C to collect mitochondria. The pellets were treated with 15% (w/v) trichloroacetic acid for 30 min, centrifuged at 3,000 X g for 1 min, and dissolved in two-dimensional gel lysis buffer (“mitochondria”). The supernatants from the mitochondrial preparations were then centrifuged at 150,000 X g in a tabletop ultracentrifuge (Beckman) for 75 min. The resulting pellets were treated with 15% (w/v) trichloroacetic acid for 30 min, centrifuged at 3,000 X g for 1 min, and dissolved in two-dimensional gel lysis buffer (“membranes”). The supernatants were transferred to new microcentrifuge tubes, and 100% (w/v) trichloroacetic acid was added to give a final concentration of 15% (w/v). After incubation for 30 min on ice, precipitated proteins were collected by centrifugation at 3,000 X g for 5 min and dissolved in two-dimensional gel buffer (“cytosol”). For“whole cell lysate,” 100 p1 of the original sample was removed, the cellular material precipitated with a final concentration of 15% (w/v) trichloroacetic acid for 30 min on ice, and then EXPERIMENTALPROCEDURES centrifuged at 3,000 X g for 1 min. The pellets were then dissolved in two-dimensional gel lysis buffer. Tissue Culture-HIR 3.5 cells (28) and mouse C127 cells stably I n Vitro Phosphorylation-Confluent quiescent cultures of HIR expressing more than IO6 human insulin receptors/cell were kindly 3.5 cells in two 100-mm dishes were incubated overnight in serumprovided by Dr. Jonathan Whittaker (SUNY, Stony Brook). The free DMEM containing 1% (w/v) bovine serum albumin. The cells cells were grown in a humidified 5% CO,, 95% air incubator at 37 ‘C were washed three times in ice-cold phosphate-buffered saline conin Dulbecco’s modified Eagle’s medium (DMEM; GIBCO) containing taining 35 p M P A 0 and then harvested by scraping in 1 ml of the 25 mM D-ghCOse, 100 units/ml penicillin, 100 pg/ml streptomycin, same solution. The cells were quickly pelleted by centrifugation at and 10% (v/v) heat-inactivated iron-supplemented calf serum 1,000 X g for 3 min at 4 “C and then resuspended in lysis buffer (50 (HyClone Laboratories, Inc., Logan, UT). mM Tris/HCl 7.4), 0.5 mM EDTA, 0.5 mM EGTA, 35 p M PAO, Labeling and Treatment of Cells-HIR 3.5 cells were grown to 0.25 M sucrose,(pH 1% (w/v) Triton X-100). The lysate was passed 10 confluence in 35-mm dishes. The day before the experiment, cells times through a 26-gauge needle and then incubated on ice for 45 were refed with DMEM in which 1%(w/v) bovine serum albumin min. Insoluble material was pelleted by centrifugation a t 200,000 X g (bovine serum albumin; 5 X recrystallized, essentially fatty acid free, in a tabletop ultracentrifuge for 20 min at 4 “C.The supernatant was Sigma)had been substituted for serum. After 18 h in serum-free transferred into 2 microcentrifuge tubes, and either water or insulin medium, cells were washed four times with phosphate-free DMEM (1p M ) was added. After 10 min at 25 “C (toallow the insulin to hind and labeled for 1-2 h in 0.6 ml of phosphate-free DMEM (GIBCO) to itsreceptor), the lysate was adjusted to contain10 mM MnC1?,200 containing 25 mM HEPES, pH 7.3, and 2 mM L-glutamine, 1% (w/v) p~ ATP (Sigma), and 1.5 pCi of [Y-~’P]ATP(crude, 7,000 Ci/mmol; bovine serum albumin, and 1 mCi/ml [32P]orthophosphate (DuPont- ICN Radiochemicals, Irvine, CA) and incubated for a further 20 min New England Nuclear). Ten min before the addition of hormones or at 25 “C. The reaction was stopped by the addition of 200 p1 of icegrowth factors, P A 0 or vehicle (dimethyl sulfoxide) was added to a cold 25% (w/v) trichloroacetic acid. After 30 min on ice, the precipifinalconcentration of 35 p~ (PAO)and/or 0.1% (v/v)dimethyl tate was collected by centrifugation for 5min at 2,000 X g, the sulfoxide. Insulin(70 nM; Lilly), fetal bovine serum, phorbol 12- supernatant removed, and the pellet lysed in 100 pl of two-dimenmyristate 13-acetate (1.6 p ~ Sigma), ; platelet-derived growth factor sional gel sample buffer. Aliquots of the lysates were matched for (PDGF, 10 ng/ml;Amgen Biological, Thousand Oaks, CA), fibroblast radioactivity and analyzed by giant two-dimensional gel electrophogrowth factor (FGF, 1ng/ml; Amgen), epidermal growth factor (EGF, resis. 10 nM; Collaborative Research, Bedford, MA), or insulin-like growth Quantitation-The amount of label incorporated into [3ZP]orthofactor 1 (IGF-1, 10 nM; Amgen) was added for a further 1-25 min, phosphate-labeled proteins was determined by densitometry of twoafter which the dishes were placed on ice and washed three times dimensional autoradiographs using a Hoefer GS300 scanning densiwith ice-cold DMEM. The cells were solubilized in two-dimensional tometer (HoeferScientific Instruments, San Francisco, CA), caligel lysis buffer, and protein concentration and trichloroacetic acidbrated with a Kodak optical density calibrationstrip. precipitable radioactivity were determined as described (30). Cells Phosphoarnino Acid Analysis-Proteins were eluted from gels as were labeled with [3sS]methionine as described previously (30). described (31), and phosphoamino acid determination was performed Two-dimensional Gel Electrophoresis-Cell lysates containing 0.5as described (32). 2 million trichloroacetic acid-precipitable cpm (32P) or5-20 million Anti-phosphotyrosine Antibody Affinity Chromatography-HIR 3.5 cpm (3sS)were separated using giant two-dimensional gel electrophoresis as described (30). The gels were fixed, dried, and exposed to cells were grown in 100-mm plates (one plate/condition), labeled with [32P]orthophosphate, pretreatedwith PAO, and then treatedwith or Kodak XAR film with one intensifying screen at -70 “C for 12-96 h without insulin as described above. A commercially available anti(”P) or 2-5 days without a screen at room temperature (3sS).Within phosphotyrosine antibody (clone 1G2, developed by A. R. Frackelton, an experiment, equal amounts of trichloroaceticacid-precipitable radioactivity from each experimental dish were applied to the first- Brown University (33)) coupled to Sepharose beads (Oncogene Sciences, Manhasset, NY) was used to isolate labeled proteins presumdimension gels. ably containing phosphotyrosine. All steps were performed exactly as recommended by the manufacturer, using the lysis, wash, elution H. B. Sadowski, R. M. Levenson, and D. A. Young, unpublished (with the hapten phenyl phosphate), regeneration, and storage buffers data. provided. To aid inrecovery of the sample, 100 pg of nonradioactive
P A 0 inhibiteda phosphotyrosine phosphatase activity in these cells. The authors proposed that an insulin-stimulated transient tyrosine phosphorylation of the 15-kDa protein had been stabilized by PA0 (26, 27). With the exception of the insulin receptor itself, the 15-kDa protein was the only substrate phosphorylated by insulin in the presence of PA0 in the 3T3-Ll adipocytes, and its phosphorylation on tyrosine suggested that it might be a direct substrate for the insulin receptor protein tyrosine kinase. To discover whether other cryptic putative substrates for the insulin receptor exist, we utilized an NIH 3T3 cell line expressing high levels of stably transfected human insulin receptors (28) and examined their protein phosphorylation responses to insulin in both the absence and presence of PAO. We separated cellular proteins using an ultra-high resolution “giant” two-dimensional gel electrophoresis system that can resolve more than 1200 32P-labeled phosphoproteinsin cellular lysates (29).*We found that in the absence of PAO, more than 25 proteins were phosphorylated in response to insulin. In cells pretreated with PAO, insulin specifically stimulated the phosphorylations of at least 26 proteins, of which at least 10 were phosphorylated on tyrosine residues. Only one of these PAO-insulin-phosphoproteins (PIPs) overlapped with the insulin-stimulated phosphorylations seen in the absence of PAO. It is possible that at least some of these PIPs are direct substrates for the insulin receptor tyrosine kinase and as such may play a role in mediating the actions of insulin.
Insulin-stimulated Tyrosine Phosphorylation
19986
cellular protein was added to theelution buffer before the addition of 100% (w/v) trichloroacetic acid to give a final trichloroacetic acid concentration of 15%.After incubation on ice for30 min, the samples were centrifuged for 5 min a t 2000 X g. The resulting pellets were washed with 100% ethanol and heated in 100 pl of sodium dodecyl sulfate (SDS) sample buffer (1% (w/v) SDS, 0.083 M dithiothreitol, 0.25 M sucrose, 0.01 M EDTA) at 100 “Cfor 3 min. Twenty-pl aliquots were then separated on prepoured 4-20% acrylamide gradient SDSpolyacrylamide gel electrophoresis minigels (Novex, Encinitas, CA) for 1 h. The gels were dried and exposed to Kodak XAR film with intensifying screen overnight.
In Fig. 1, two proteins are enclosed in boxes. One, labeled W, was dephosphorylatedby insulin; the other, V, is the most basicformof the 21-22 kDa phosphoprotein described in 3T3-Ll mouse fibroblasts and adipocytes(34-36). Insulin causes the addition of further phosphate groups to V, and the apparent dephosphorylation is probably dueto a shift of the more acidicphosphorylated protein toward the anode, i.e. off the gels to the right (34-36). Twenty-four of the 26 proteins phosphorylated by insulin were also phosphorylated by serum and to approximately the same degree. The two exceptions were: 1) the 95-kDa pRESULTS subunit of the insulin receptor itself ( I R ) ,and 2) protein f Protein Phosphorylations inthe Absence of PAO-In order (identified with an open arrowhead), which will be discussed to identify proteins that were rapidly phosphorylated follow- below. ing insulin treatment, we prelabeled HIR 3.5 cells with [”PI Serum also causedthe phosphorylation of several proteins orthophosphate and then incubated the cells with insulin or (not circled, but indicated with closed arrowheads) that were fetal bovine serum for various periods of time. Whole cell not insulin responsive. Studies performed in Swiss 3T3 cells lysates were prepared and the proteins separated on giant suggested that many of these phosphoproteins are direct two-dimensional gels as described under “Experimental Procedures.” Fig. 1 shows representative gels from cells treated substrates for protein kinase C2and were always foundto be with vehicle alone ( A ) ,insulin ( B ) ,or fetal bovine serum (C) phosphorylated in tandem with the “80K” or MARCKS profor 10 min. Of these phosphoproteins, at least 26 were phos- tein, a prominent, apparently specific, marker for protein phorylated and 1dephosphorylatedby insulin within the first kinase C activation in intact cells (35,37-39). The MARCKS 10 minof treatment. Theproteins phosphorylated by insulin protein, indicated by an open diamond inpanel B, is located are circled and identified with capital letters or with IR (8- at the acidic end of the autoradiographs; shorter exposures subunit of the human insulin receptor) or EF-2 (elongation demonstrate serum- but not insulin-dependent phosphorylafactor 2 (30)). The &subunit of the human insulin receptor tion of this protein (data not shown). The inability of insulin was identified onthe basis of its M,(95,000), isoelectricpoint to stimulate the phosphorylation of either the MARCKS (5.1),rapid and specific phosphorylation following insulin protein or other protein kinase C-sensitive marker proteins treatment, and itsappearance in the HIR 3.5 cell line, which indicates that the hormone does not activate protein kinase overexpresses the insulin receptor, and not in the parental C in these cells. This conclusion is supported by our demonNIH 3T3 cell line (data not shown). Estimated molecular stration that in these cells, insulin also fails to stimulate masses and isoelectric points for the insulin-stimulated phos- membrane inositol phospholipid turnover, which is known to phorylations are given in Table I along with identification of lead to protein kinase C activation (data not shown). Proteins Phosphorylated in the Presence of PAO-In order the amino acid residuesthat were phosphorylated, when that was determined. to determine whether additional insulin-stimulated phosB
4s
-
31
-
21
-
14
boslc
Insulin
acidic
FIG. 1. Effect of insulin and serum on protein phosphorylationsin HIR 3.5 cells. Confluent quiescent cultures of HIR 3.5 cells were prelabeled with [32P]orthophosphatefor 2 h and thentreated with DMEM (control, panel A ) , insulin (70 nM, panel B ) , or 15% (v/v) fetal bovine serum (panel C) for 10 min. Whole cell lysates were prepared and separated on giant two-dimensional gels as described under “Experimental Procedures.” Gels were dried and exposed to Kodak XAR filmovernight at -70 “Cin the presence of an intensifying screen. In B, proteins whose phosphorylation was stimulated by insulin are circled and identified with capital letters or with ZR, EF-2, or f (a stimulated phosphorylation also seen in the presence of PAO; see Fig. 2 and “Results”). Two proteins are enclosed in boxes. One, termed, W , is dephosphorylated by insulin; the other, V, is the basic form of a 21-22-kDa phosphoprotein that is phosphorylated further by insulin (see “Results”). The corresponding proteins from control cells are indicated in A. In C, 24 of the 26 proteins whose phosphorylation was stimulated by insulin were also phosphorylated in response to serum. The two exceptions were 1) the 95-kDa &subunit of the insulin receptor itself ( I R ) ;and 2) protein f (identified with an open arrowhead). Serum also caused the phosphorylation of proteins (indicated with closed arrowheads) or with an open diumond in panel B, the 80 K or MARCKS protein that were not phosphorylated in response to insulin (see “Results”).
Phosphorylation Insulin-stimulated Tyrosine TABLEI Molecular masses, isoelectric points, and phosphoaminoacid analysis of proteins phosphorylated by insulinin the absence of P A 0 Spot names,apparentmolecular masses, andisoelectric points correspond to the phosphoproteinsshown in Fig. 1. The labeled phosphoaminoacidresidues were determined as describedunder “Experimental Procedures.” Spot name
M$i:lar PI
Phosphoamino acid
kDa
X
95 96 70 66 57 56 51 51 50 50 46 44 43 43 43 42 40 40 39 38 35 31 28 26 23 23 19
5.10 6.95 5.65 6.50 5.80 5.60 7.50 7.30 5.30 5.00 6.50 5.70 6.50 6.20 6.40 5.50 6.65 6.50 6.15 6.90 6.45 5.80 5.85 5.20 5.00 6.45 5.90
Y
18
5.20
IR EF-2 A B C D E F G H I J K L M N 0 P
Q
f
R S T
U V W
P-Tyrp P-Ser P-Thr, P-Ser
P-Tyr, P-Ser, P-Thr P-Ser P-Ser, P-Thrb (Dephosphorylated) P-Ser (determined in Swiss 3T3 cells)
P-Tyr, phosphotyrosine;P-Ser, phosphoserine;P-Thr, phosphothreonine. Ref. 35.
phorylations could be demonstrated following insulin stimulation in the presence of PAO, we prelabeled HIR 3.5 cells with [32P]orthophosphate,treated the cells for 10 min with P A 0 (35 p M ) , and then incubated the cells with insulin or fetal bovine serum, still in the presence of PAO, for various periods of time. Autoradiographs from a representativeexperiment (from a series of seven) are shown in Fig. 2. Although the overall pattern of phosphorylations inunstimulated cells is similar in the absence and presence of PAO, treatment with this agent caused a marked decrease in the phosphorylation of a number of individual phosphoproteins (compare panel A , Figs. 1 and 2). For example, PA0 caused the complete dephosphorylation of EF-2, which is prominent in Fig. 1 and no longer visible in Fig. 2 or in immunoprecipitates isolated with anti-EF-2 antiserum (data not shown). Also note that the cluster of phosphoproteins immediately above protein f and the dark spot to the left of protein X in Fig. 1 are missing in Fig. 2. On the other hand, there areno convincing novel phosphorylations evidentin PAO-pretreated control cells. However, when insulin was added to PAO-pretreated cells, more than 26 proteinphosphorylations were stimulated. These proteins are circled and labeled with small letters in Fig. 2B, and theirapproximate molecular masses and PIvalues reveals a novel are listed in Table11. Thus, PA0 pretreatment
19987 set of insulin-sensitive phosphorylations (PIPs). With only two exceptions, there was no overlap between insulin-stimulated phosphorylations seenin thepresence and absence of PAO. The exceptions were the P-subunit of the insulin receptor, and protein f (36 kDa, p~ 6.9), which is shown in both Figs. 1 and 2. Unimpeded autophosphorylation of the insulin receptor in the presence of PA0 has been documented previously (26). Serum, concentrations in sufficiently high enough to stimulatenumerousproteinphosphoryiatiois in no;-PAO-treated cells (Fig. l),did not stimulate the phosphorylation of any proteins in thepresence of PA0 (compare Fig. 2, A and C). Phosphoamino Acid An~lysis-[~~P]Orthophosphate-labeled cell lysates from PAO-pretreated insulin-treated cells were separated on two-dimensional gels, and phosphoamino acid analysis was performed on 10 of the more abundant PIPs. As shown in Fig. 3 and in Table 11, all 10 of these PIPs contained phosphotyrosine predominantly or exclusively. In contrast, when we examined phosphoamino acid residues on several proteins labeled in the absence of PAO, with the exception of the p-subunit of the insulin receptor, only phosphoserine or phosphothreonine was detected (Table I). AS expected, the @-subunit of the insulin receptor contained mostly phosphoserine and phosphotyrosine residues, and EF2 contained mostly phosphothreonine with a small amount of phosphoserine (40). In Vitro Phosphorylation-Since many of the PIPs contained phosphotyrosine and thus might be direct substrates for the insulin receptor protein kinase, we investigated whether they could be phosphorylated in crude cellular extracts of HIR 3.5 cells stimulated in vitro with insulin in the presence of [Y-~’P]ATP,Mn2+ ions, and PAO. Addition of insulin to thiscell-free system stimulatedthe phosphorylation of several proteins, including the &subunit of the insulin receptor and seven of the PIPs seen previously in insulintreated intact cells (Fig. 4). Autophosphorylation of the insulin receptor and insulinstimulated kinase activity directed toward other substrates are supported better by Mn2+ than by M$+ (3, 31, 41, 42). When an in vitro phosphorylation assay was performed in the presence ofMg2+ rather than Mn2+, no insulin-stimulated phosphorylations (except the insulin receptor)were seen (data not shown). Time Course of Phosphorylation-When cells were exposed to insulin for various times in the absence and presence of PAO, we found that theinsulin receptor appeared fully phosphorylated within 1 min of insulin treatment, whereas all other phosphorylations were maximal by 10 min and did not change appreciably over the next 15 min (data not shown). Although none of the phosphorylations seen in the absence of P A 0 was evident at the 1-mintime point (with the exception of protein f), the insulin-stimulated phosphorylation of all the PIPs was readily detectable by 1 min (Fig. 5). The more rapid phosphorylation of the PIPs supports the possibility that they, unlike the proteins phosphorylated in response to insulinin the absence of PAO,may be direct substrates for the insulin receptor. Not shown in panel A is the time course of the phosphorylation of protein f in the absence of PAO. With the exception of the insulin receptor, this insulin-stimulated phosphotyrosine-containing protein was the only phosphorylation seen in both the absence and presence of PAO, in both cases by 1 min. This is consistent with the finding noted above (Fig. 1) that, in the absence of PAO, it was one of only two proteins whose phosphorylation was stimulated by insulin but not by serum. Phosphorylation in Other Cell Types-Bernier and co-work-
Insulin-stimulated Tyrosine
19988
Phosphorylation C . Serum (+) P A 0
B. Insulin (+) P A 0
A Control 1+1 P A 0
4 95-
66-
45-
31-
2114-
ocldlc
baslc
FIG. 2. Effect of insulin and serum on protein phosphorylations in HIR 3.5 cells pretreated with phenylarsine oxide. Cells were prelabeled and treated with agonists and analyzed as described in Fig. 1, except ) added 10 min before the addition of insulin or serum and was also added to the cell lysis that PA0 (35 p ~ was buffer. In B are shown 26 proteins whose phosphorylation was stimulated by insulin (circled and labeled with small letters). With two exceptions (the &subunit of the insulin receptor and protein f), there was no overlap between the insulin-stimulated phosphorylations seen in the presence (Fig. 2B) and absence (Fig. 1B) of PAO. In C is shown the lack of effect of serum on the proteins whose phosphorylation was stimulated by insulin (compare with 28).
TABLE I1
P-Ser
Molecular masses, isoelectric points, and phosphoaminoacid analysis of P A 0 of proteins phosphorylated by insulin in the presence Spot names, apparent molecular masses, and isoelectric points correspond to the phosphoproteins shown inFig. 2. The labeled phosphoamino acid residues were determined as described under “Experimental Procedures.” Spot name
mass
PI
Phosphoamino acid
kDa
IR a 5.70 C 6.15 d 6.45 e f g 6.65 h 5.90 I 5.75 1 6.00 m n 0
P 9
95 50 48 47 39 38 37 37 37 33 33 30 28 28 28 26 26 25 24 24 20 19 16 16 15 12
5.10
6.45 6.90
7.20 5.85 6.40 5.85 6.00 7.10 5.30
P-Tyr,” P-Ser
P-Tyr, P-Ser, P-Thr P-Tyr
P-Tyr, P-Ser P-Tyr P-Tyr P-Tyr
P-Thr
P- Tyr
f
g
p
k
I
o
t
y
L
w
Ins.
EF-2
Rec.
FIG. 3. Phosphoamino acid analysis ofproteins whose phosphorylation was stimulated by insulin in the presence of PAO. Ten of the most prominent insulin-stimulated phosphoproteins were eluted from gels similar to that shown in Fig. 2B, whereas the 0subunit of the insulin receptor and EF-2were eluted from gels similar to thatshown in Fig. 1B. The phosphoproteins were hydrolyzed in 6 N HCI, and the labeled phosphoamino acids were separated by thinlayer electrophoresis as described under “Experimental Procedures.” The locations of phosphoserine (P-Ser),phosphothreonine (P-Thr), and phosphotyrosine ( P - T y r )were determined with unlabeled standards visualized with ninhydrin. Lower case letters denote phosphoproteins whose locations are given in Fig. 2B. All 10 proteins from the PAO-treated cells were labeled predominantly or exclusively on tyrosine residues. The insulin receptor (Ins.Rec.) contained phosphoserine and phosphotyrosine, and EF-2 predominantly phosphothreonine, as reported previously (4,40).
the 15-kDa protein reported previouslyby Lane and coworkers (23,27,43). P-Tyr 6.15 We also examined a mouse C127 cell line transfected with U 6.70 a cDNA for the human insulin receptor (provided by Dr. V 6.40 Jonathan Whittaker). C127 cells are derived from a mouse W P-Tyr, P-Ser 5.40 mammarycarcinoma (44) and are thus likely to beof a X 6.00 different embryological origin than HIR 3.5 cells. When in6.50 P-Tyr Y sulin-sensitivephosphorylations in the presence of PA0 were P-Tvr Z 6.20 P-Tyr, phosphotyrosine; P-Ser, phosphoserine; P-Thr, phospho- determined in this cell line, we found that at least 11 of the HIR 3.5 PIPS were also present in the C127 cells (proteins b, threonine. f, g, 1, p, q, t, u, v, w, z; data not shown). Specificity-To determine the insulin specificity of these ers (23) reported that insulin stimulated the phosphorylation of a 15-kDa protein in PAO-pretreated 3T3-Ll adipocytes. phosphorylations, we treated HIR 3.5 cells prelabeled with We also found only one protein whose phosphorylation was [32P]orthophosphateand PA0 with PDGF, EGF, FGF, IGFstimulated by insulin in the presence of PA0 in these cells 1, insulin, or vehicle alone for10min and separated the (Fig. 6). It co-migrated with protein y in Fig. 2B, had an M, phosphoproteins on two-dimensionalgels as described above. of 15,000 and a PI of about 6.5, and probably corresponds to The results are shown in Fig. 7. Only insulin stimulated PIP r 5.25 S 5.80 t
Insulin-stimulated Tyrosine Phosphorylation
-
19989
A Conlrol
M
A
-7q
95-
66-
4s-
31-
2114-
FIG. 4. Effect of insulinon protein phosphorylation ina cell-free system. Confluent, quiescent cultures of HIR 3.5 cells were harvested and lysed in a buffer containing 1%Triton X-100 as described under "Experimental Procedures." Particulate matter wasremoved by centrifugation, and the supernatant was separated into two M) was added. After 10 min, the lysates aliquots to which either water ( A ) or insulin ( E ; final concentration were adjusted to contain 10 mM MnCI2;200 p M ATP, and 1.5 pCi [y-32P]ATPin a final volume of 200 pl. After 20 min at 25 "C, the reactions were stopped and the phosphoproteins separated on giant two-dimensional gel electrophoresis. Insulin-stimulated protein phosphorylations seen in common with those occurring in uiuo in the presence of PA0 (Fig. 2 8 ) are labeled with small letters. Additional insulin-stimulated phosphorylations not seen in vivo are circled without labels.
C. Averages
B. (+) PA0
A. (-) PA0
I
2.0
ti? z w
0
1.5
k
-
: ;1.0n
0
0.5
0
1
10
0
1
10
0
1
10
(MINUTES) (MINUTES) TIME (MINUTES) TIME TIME
FIG. 5. Time course of insulin-stimulated protein phosphorylations occurring in the absence or presence of PAO. HIR 3.5 cells were prelabeled with [32P]orthophosphatefor 2 h, pretreatedwith PA0 (35 p M ) or vehicle alone for 10 min, and then treated with insulin for 1, 5, 10, 15, 20, and 25 min. Whole cell lysates were then separated on two-dimensional gels. The insulin receptor was fully phosphorylated within 1 min of insulin treatment; all other insulin-dependent phosphorylations were maximal by 10 min and did not change appreciably over the next 15 min (data not shown). Autoradiographs of cells treated for 0, 1, and 10 min were analyzed with a scanning densitometer, and the optical densities of 10 prominent insulin-stimulated phosphoproteins (a different set for each condition) from untreated ( A ) and PAO-treated ( E ) cells were determined. In A, little or no insulinstimulated protein phosphorylation occurred within the first min, whereas all the proteins were markedly phosphorylated by 10 min. In E , phosphorylation of all the proteins was measurably increased after 1 min of insulin treatment, although further increases occurred during the next 9 min of incubation. In C, the means (& S.E.) of the densities of all 10 spots in panels A and 8 are plotted.
Abundance-Protein t, the most prominent PIP, could be phosphorylation in the presence of PAO; the other growth factors, with the possible exception of PDGF, were inactive. readily demonstrated by silver staining of the gels: however, This is in agreement with results obtained by Bernier et al. none of the other PIPs could be detected in silver stains of (23) with respect to the15-kDa protein alone. This finding is gels fromunfractionated whole cell lysates (data not shown). also consistent with our negative results with serum in the When a protein is phosphorylated, the phosphate groups presence of PA0 (see Fig. 2). These growth factors were also confer additional negativecharges, altering its isoelectric location on two-dimensional gels. Six ineffective at stimulating PIP phosphorylation in serum-de- point andthusits proteins from [35S]methionine-labeled cells shifted into locaprived PAO-treated Swiss 3T3 cells (data not shown). SubcellularLocalization-Most of the PIPs(17of 26) were tions corresponding to six of the PIPs( f ,g, l, 0,p , and t) after present in a cytoplasmic cellular fraction (Fig. 8). There were 10 min of exposure to insulin (Fig. 9). We cannot identify alsoseveral phosphorylations stimulated by insulin which with certainty the origins of these "shifted" proteins:it therewere detectable in the cytoplasmic fraction but not in whole fore remains possible that they appearedin the cytosolic cell lysates (circled, withoutletters). Seven proteins ( j , p , t, u, fraction because of dislodgingfrom a particulate fraction, w ,y, and z ) as well as a handful of other unnamed PIPs, were although we have no direct evidence to support this possibilprotein shifts could detected in membrane (Fig. 8), mitochondrial, and nuclear ity. The PIPsfor which no corresponding be demonstrated may not label wellwith [35S]methionine, fractions (not shown).
Insulin-stimulated Tyrosine Phosphorylation
19990
A. Control M,x10.3
FIG.6. Effect of insulin on protein phosphorylation in PAOtreated 3T3-Ll adipocytes. Confluent cultures of 3T3-Ll adipocytes were prelabeled with [RzP]orthophosphate, pretreated with PAO, and treated with control conditions ( A ) or insulin (I?) as described under "Experimental Procedures." Whole cell lysates were then analyzed on two-dimensional gels. Only one insulin-stimulated protein phosphorylation is seen in response to insulin; it corresponds to protein y in HIR 3.5 cells (see Fig. 2B).
-
B. Insulin ~
95
66
45
31
. .
21 14
A. Control
B. Insulin
C. IGF-I
W
FIG. 7. Effect of various growth factors on protein phosphorylation in PAO-pretreated HIR 3.5 cells. HIR 3.5 cells were prelabeled with ["'PI orthophosphate, pretreated with PAO, and treated with insulin and growth factors for 15 min as described under "Experimental Procedures." Whole cell lysates were then analyzed on two-dimensional gels, and portions of the resulting autoradiographs are shown. A, control; R,insulin (70 nM); C,IGF-1 (10 nM); D, EGF (10 nM); E , FGF (1 ng/ml); G, PDGF (10 ng/ml). Only insulin, and to a limited degree, PDGF, stimulated protein phosphorylations in the PAO-pretreated cells.
may be present in extremely low amounts, or may be phosphorylated with too low a stoichiometry to cause a visible shift in the gels. Relationship between Phosphoproteins Detected on Twodimensional Gels and Those Bound by an Anti-phosphotyrosine Antibody Affinity Matrix-PAO-pretreated, control, and insulin-treated HIR 3.5 cell lysates were applied to an antiphosphotyrosine antibody affinity column (33). Bound proteins were then eluted and subjected to one- and two-dimensional gel electrophoresis (Fig. 10). Most phosphorylated proteins thatadhered to thecolumn and were eluted with phenyl phosphate had an MI of greater than 30,000. In contrast, on two-dimensional gel autoradiographs, the majority of the PIPs had MI less than 30,000 (see Fig. 2 and Table 11). With the exception of the insulin receptor &subunit, none of the material eluted off the anti-phosphotyrosine antibody column and analyzed on two-dimensional gels co-migrated with any of the PIPs (data not shown). DISCUSSION
In this report, we describe several novel proteins whose phosphorylation on tyrosine was stimulated by insulinin intact HIR 3.5 cells. Three experimental features were probably important in revealing the existence of these proteins. First, we used a very insulin-sensitive cell line, the HIR 3.5, which expresses more than IO6 normal human insulin receptors/cell (28). Second, the cells were pretreated with phenylarsine oxide, a trivalent arsenical that may act to inhibit a phosphotyrosine phosphatase activity (27). Third, we ana-
lyzed the cellular protein phosphorylations using ultra-high resolution, giant two-dimensional gel electrophoresis (29). This approach identified many phosphoproteinsnot detected using chromatography with anti-phosphotyrosine antibodies. Using this approach, we found at least 26 proteins whose phosphorylation was stimulated incells exposed to insulin for 10 min. In at least 10 of these, the phosphorylation occurred predominantly or exclusively on tyrosine residues. As suggested by Bernier et al. (27), it may be that PA0 treatment reveals insulin-stimulated phosphorylationsthat arenormally inapparent due to the action of a PAO-sensitive phosphatase. We have termed these proteins PIPs. Several lines of evidence indicate that PIPs may be direct substrates for the insulin receptor protein tyrosine kinase. First, it appears that many, if not all, of the PIPs will be found to be phosphorylated on tyrosine residues. Second, a number of the PIPswere phosphorylated in vitro in an insulin-stimulated manner in a detergentextract of HIR 3.5 cells containing PAO, Mn2+,and [y3'P]ATP. Under these conditions,phosphorylation was dependent on the presence of Mn2+ in the reaction mixture; MgZ+ did not support phosphorylation of the PIPs. Thisionic dependence is characteristic of the insulin receptor kinase (3, 31, 41, 42). Third, in the presence of PAO, insulin-stimulated phosphorylation of the PIPs was evident at 1 min in intact cells, whereas phosphorylation of the other proteinsseen in the absence of PA0 was evident after 10 but not after1min of insulin treatment. Finally, phosphorylation of the PIPs was insulin specific; neither serum nor the defined factors, phorbol 12-myristate 13-acetate,PDGF,EGF, FGF, or IGF-1stimulated phos-
Insulin-stimulated TyrosinePhosphorylation
19991 8 . Cytoplasm: Insulm
b -.
A. Cyloplorm: Control
M r M J
:.-
95-
6.5.
FIG. 8. Subcellular fractionation of insulin-stimulated phosphoproteins. HIR 3.5 cells were prelabeled with [:”P]orthophosphate, pretreated with PA0 (35 pM), and exposed to insulin (70 nM) for 15 min.Subcellular fractions were prepared by differential centrifugation as described under “Experimental Procedures.” All fractions were precipitated with trichloroacetic acidand resuspended in urea/Nonidet P-40lysis buffer heforebeinganalyzedon two-dimensional gels. A, cytoplasm, control. B, cytoplasm, insulin treated. Seventeen out of 26 insulin-sensitive phosphorylations seen in Fig. 2 were present in the cytoplasmic fractions as well as additional phosphorylations not seen in whole cell lysates (circled, not labeled with a letter). C, membrane, control.D, membrane, insulin treated. Seven out of the 17 proteins in the cytoplasmic fraction were also seen in the crude membrane fraction; nomembrane-specific insulin-stimulated phosphoproteins were seen.
45.
31
21
14
0.Membrone:
C. Membrane: Control
Insulin
M h 4
21
+INS
-INS
. )
200-
92-
8 @
. I ,
-
FIG.9. Effect of insulin on the electrophoretic migration of 30 [3”S]methionine-labeledproteins. HIR 3.5 cells were labeled with (‘sS]methionine overnightinserum-free low methionine medium, 21 then treated for 10 min with P A 0 (35 p M ) and a further 10 minwith insulin (70 nM). Six new proteins appeared in locations corresponding 14 to the positions of the insulin-sensitive phosphoproteins f, g, 1, 0, p , and t (see “Results”), suggesting that a phosphorylation-dependent FIG.10. Binding and elution of phosphoproteins from an shift in isoelectric point had occurred. anti-phosphotyrosine antibody affinity column. HIR 3.5 cells were prelabeled with [”P]orthophosphate, pretreated with P A 0 (35 phorylation of any PIPin the presence of PAO. Althoughthis PM) and exposed to insulin (70 nM) for 15 min. Cell lysates were prepared from insulin- (+INS)and control-treated (-INS), matched circumstantial evidence supports the contention that the PIPS for trichloroaceticacid-precipitable cpm,and applied to an antiare substratesof the insulin receptor protein tyrosine kinase, phosphotyrosine antibody column as described under “Experimental proof of this contention will require phosphorylationsite Procedures.” Phosphoproteins remaining on the column after washanalysis from intact cell experiments as well as in uitro studies ing were eluted with phenyl phosphate, precipitated with trichloroacetic acid, and analyzed by SDS-gel electrophoresis and autoradiogusing purified kinase and substrates. The mechanism by which P A 0 exerts its biological effects raphy, shown here. The positions of molecular weight standards are shown.
-
is known in general, but the specific target in HIR 3.5 cells with which it interacts to modify insulin-stimulated protein phosphorylation-dephosphorylation reactions remains to be defined.Arseniccompounds inhibitenzymaticactivity by binding reversibly to vicinal dithiols such as sulfhydryl groups foundin proteins (24, 45).Despite the wide spectrum of inhibited enzymes (reviewed in 46), cells exposed in culture to sublethal but cytostaticdoses of arsenic for 24 h were still
capable of normal growth after treatment with an antidote, 2,3-dimercaptopropanol(46).Furthermore, P A 0 a ta concentration of 18 PM had only modest effects on cellular ATP level in 3T3-Ll adipocytes, causing a 10-20% decline over 30 min (25).However, results of experiments inwhich this agent is used should be interpreted with caution since P A 0 inhibits
19992
Phosphorylation Insulin-stimulated Tyrosine
basal (47) and/or insulin-stimulated glucose uptake (25), pro- HIR 3.5 cells from Dr. Jonathan Whittaker and the excellent techtein synthesis, and phosphate uptake: and perhaps other nical assistance of Dale MacNeill Haupt with giant two-dimensional gel electrophoresis. We also thank Dr. Graham Carpenter for comphysiological processes. Nevertheless, it does not appear to municating results before their publication and Drs. M. Daniel Lane inhibit the insulin receptor kinase itself and permits the and A. Raymond Frackelton for helpful discussions. We acknowledge visualization of novel, insulin-stimulated protein phos- the helpful comments of our colleagues, Drs. Jonathan Graff, David phorylations in intact cell studies. M. Harlan, and Michael W. Roe. None of the other growth factors tested, including several REFERENCES whose receptors are protein tyrosinekinases, had asignificant 1. Jacobs, S., and Cuatrecasas, P. (1981) Endocr. Rev. 2, 251-263 effect on the phosphorylation of the PIPs. It may be that 2. Rosen, 0. M. (1987) Science 237, 1452-1458 these other kinases, unlike the insulin receptor kinase, are 3. Avruch, J., Nemenoff, R. A., Blackshear, P. J., Pierce, M. W., inhibited directly by PAO. Alternatively, the insulin receptor and Osathanondh, R. (1982) J. Biol. Chem. 257, 15162-15166 kinase may phosphorylate substrates with unique specificity. 4. Kasuga, M., Karlsson, F.A., and Kahn, C.R. (1982) Science An example of such specificity has recently been demon215,185-187 strated by Nishibe et al. (48),who found that the insulin 5. Kasuga, M., Zick, Y., Blith, D. L., Karlsson, F. A., Haring, H. U., and Kahn, C. R. (1982) J. Biol. Chem. 257,9891-9894 receptor was incapable of phosphorylating a subtypeof phos6. Kasuga, M., Zick, Y., Blithe, D. L., Crettaz, M., and Kahn, C. R. pholipase C which was readily phosphorylated by the EGF (1982) Nature 298,667-669 and PDGF receptor kinases. Although the tyrosine kinase Petruzzelli, L. M., and 7. Rosen, 0. M., Herrera, R.,Olowe,Y., domain of the insulin receptor bears a strong similarity to Cobb, M. H. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 3237those of the other receptor tyrosine kinases (9), additional 3240 regions of the insulin receptor appear to convey substrate 8. White, M. F., Shoelson, S. E., Keutmann, H., and Kahn, C . R. (1988) J. Biol. Chem. 263,2969-2980 specificity since substitution of tyrosine 960 with phenylala9. Ebina, Y.,Ellis, L., Jarnagin, K., Edery, M., Graf, L., Clauser, E., nine blocks phosphorylation of substrates in intact cells withOu, J.-H., Masiarz, F., Kan, Y. W., Goldfine, I. D., Roth, R. A., out affecting autophosphorylation or phosphotransferase acand Rutter, W. J. (1985) Cell 40,747-758 tivity in vitro (49). It is possible that the insulin receptor 10. Ellis, L., Clauser, E., Morgan, D., Edery, M., Roth, R. A., and kinase phosphorylates a unique class of substrates with a Rutter, W. J. (1986) Cell 45, 721-732 specificity distinct from other known kinases; these phos- 11. Chou, C . K., Dull, T. J., Russell, D. S., Gherzi, R., Lebwohl, D., phorylated proteins then become potential substrates for a Ullrich, A., and Rosen, 0. M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,704-708 tyrosine phosphatase activity that is inhibitable by PAO. The PIPs may have escaped detection until now because 12. Chou, C. K., Dull, T. J., Russell, D. S., Gherzi, R., Lebwohl, D., Ullrich, A., and Rosen, 0. M. (1987) J. Biol. Chem. 262,1842they appear not t o bind to ananti-phosphotyrosine antibody 1847 with appreciable avidity. When anti-phosphotyrosine anti- 13. Stumpo, D. J., Stewart, T. N., Gilman, M. Z., and Blackshear, P. bodies have been used to detect phosphotyrosine-containing J. (1988) J. Biol. Chem. 263, 1611-1614 proteins, either in insulin-treated cells (this report and Ref. 14. White, M. F., Maron, R., and Kahn, C . R. (1985) Nature 318, 183-186 22) or in cells transformed by a range of viral oncogenes expressing tyrosine kinase activity (50), the majority of pro- 15. Izumi, T., White, M. F., Kadowaki, T., Takaku, F., Akanuma, Y., and Kasuga, M. (1987) J. Biol. Chem. 262, 1282-1287 teins detected had relatively high molecular mass (90% with 16. Kadowaki, T., Koyasu, S., Nishida, E., Tobe, K., Izumi, T., molecular mass greater than 50 kDa and 40% greater than 90 Takaku, F., Sakai, H., Yahara, I., and Kasuga, M. (1987) J. kDa (50)). This is in contrast to the size distribution of the Biol. Chem. 262,7342-7350 PIPs, most of which are 28 kDa or smaller. None of the PIPs 17. Gibbs, E. M., Allard, W. J., and Lienhard, G. E. (1986) J. Biol. Chem. 261,16597-16603 was specifically bound to the anti-phosphotyrosine antibody column. Reasons for this include the possibility that they 18. Shemer, J., Adamo, M., Wilson, G. L., Heffez, D., Zick, Y., and LeRoith, D. (1988) J. Bwl. Chem. 262, 15476-15482 contain phosphotyrosine residue(s) inconformations not rec19. Haring, H. V., White, M. F., Machicao, F., Ermel, B., Schleicher, ognized by these antibodies or that proteins containing only E., and Obermaier, B. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, relatively few phosphotyrosine residues may not be retained 113-117 by the affinity column. In any case, these results point out 20. Perrotti, N., Accili, D., Marcus Samuels, B., Rees Jones, R. W., the usefulness of high resolution two-dimensional gels in andTaylor, S. I. (1987) Proc. Natl. Acad. Sei. U. S. A. 84, 3137-3140 detecting and characterizing this type of protein phosphoryla21. Pasquale, E. B., Maher, P. A., and Singer, S. J. (1988) J. Cell. tion event. Phy~iol.137, 146-156 It is possible that one or more of the insulin-stimulated 22. Mooney, R. A., Bordwell, K. L., Luhowskyj, S., and Casnellie, J. protein tyrosine phosphorylations we have described may play E. (1989) Endocrinology 124, 422-429 important roles in mediating insulin action. However, not all 23. Bernier, M., Laird, D.M., and Lane, M.D. (1987) Proc. Natl. phosphorylations catalyzed by the insulin receptor kinase are Acad. Sci. U. S. A. 84, 1844-1848 necessarily physiologically significant. Stimulation of fibro- 24. Stocken, L. A., and Thompson, R. H. S. (1946) Biochem J . 40, 529-535 blasts with other growth factors hasrevealed several such low stoichiometry tyrosine phosphorylations of abundant proteins 25. Frost, S. C., and Lane, M. D. (1985) J. Biol. Chem. 260, 26462652 (51). However, none of the PIPs was particularly abundant, 26. Frost. S. C.. Kohanski, R. A., and Lane, M. D. (1987) J. Bid. and several of them appeared to be phosphorylated to a Chem. 262,9872-9876 reasonable stoichiometry, as evidenced by a shift of the [35S] 27. Bernier, M., Laird, D. M., and Lane, M. D. (1988)J. Biol. Chem. methionine-labeled proteins toward the anode after insulin263. 13626-13634 stimulated tyrosine phosphorylation. In order to determine 28. Whittaker, J., Okamoto, A. K., Thys, R., Bell, G. I., Steiner, D. F., and Hofmann, C. A. (1987) Proc. Natl. Acad. Sci. U.S. A. the biological relevance of the PIPs in insulin action, their 84,5237-5241 functional identification and insulin-stimulatedfunctional 29.Young,D. A,, Voris, B. P., Maytin, E. V., and Colbert, R. A. modification need to be established. (1983) Methods Enzymol. 91,190-214 ~
Acknowledgments-We gratefully acknowledge the generous gift of R. M. Levenson, and P. J.Blackshear, unpublished data.
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