0013-7227/92/1303-1533$03.00/0 Endocrinology Copyright Q 1992 by The Endocrine
Vol. 130, No. 3 Printed in U.S.A.
Society
ROBERT
A. MOONEY
AND KATHY
Department of Pathology and Laboratory Dentistty, Rochester, New York 14642
of a
L. BORDWELL
Medicine,
University
of
Rochester School of Medicine
ABSTRACT. Insulin stimulated the tyrosine phosphorylation of a 61-kilodalton &Da) protein in rat adipocytes prelabeled for 2 h with 13*Plorthouhosnhate. Tvrosine phosphorvlation of this 61-kDa &o&in diipla$d very- similar- in&in concentration dependency to receptor autophosphorylation and tyrosine phosphorylation of a high molecular mass receptor substrata of 160 kDa. Phoaphorylation of the Bl-kDa protein was very rapid with maximum labeling attained at 30 set, paralleling that of the other two proteins. Phosphoamino acid analysis revealed that each of the insulin-responsive phosphoproteins contained phosphoserine as well as phosphotyrosine, though the ratio of two phosphoamino acids recovered from each protein differed. The 61-kDa protein yielded relatively equal proportions of phosphoserine and phosphotyrosine. In contrast, the insulin receptor yielded relatively more label on phosphotyrosine than phosphoserine, whereas label incorporated into the 160-kDa protein was
and
recovered primarily on phosphoserine. Cleveland peptide maps using either Staphylococcus aurew V8 proteinase or chymotrypsin revealed no similarities between the 61-kDa protein and the other tyrosine phosphorylated proteins. With subcellular fractionation, the 160-kDa protein was found in equal proportions in the high speed pellet (100,000 g) and supernatant. The 61kDa protein had a similar distribution to that of the 160-kDa protein but was also detected in the low speed pallet (10,000 g). The insulin receptor was localized to the low speed pellet. In summary, rat adipocytes contain an insulin-dependent phosphotyrosyl protein of 61 kDa which is distinct from the more prominent high molecular mass receptor substrate. Thii 61-kDa protein has characteristics consistent with it being a substrate for the insulin receptor tyrosine kinase. (Endocrinology 130: 1533-1538,1992)
S
strates is also essential. This may be partly due to the difficulty in identifying and studying these rare phosphorylation events. Nonetheless, several potential substrates that are phosphorylated on tyrosine residues in an insulin-dependent manner have now been recognized. White et al. (7,8) described a 185K phosphoprotein from the Fao hepatoma cell line using an antiphosphotyrosine antibody. Insulin-dependent phosphotyrosyl proteins of similar molecular mass have also been observed in other insulin-responsive tissues (g-11), including rat adipocytes (12). We and others have observed additional insulin-dependent phosphotyrosine-containing proteins in rat adipocytes including those of 60-65 kDa (12-l@, 46 kDa (13), 17 kDa (calmodulin) (16), and 15 kDa (17). The 60-65 kDa (61-kDa in our studies) insulin-dependent phosphotyrosine-containing protein is of particular interest because it has consistently been observed in rat adipocytes. The very limited knowledge of the 60-65 kDa phosphotyrosine-containing protein in rat adipocytes, however, has been obtained through immunoblotting techniques. Direct characterization of the phosphoprotein has not been reported and is the objective of this study.
UBSTANTIAL evidence links insulin receptor tyrosine kinase activity to insulin signal transduction and insulin action. Most convincing of this evidence comes from the molecular biological approach. Cell lines transfected with human insulin receptor complementary DNA show increased insulin binding and increased sensitivity to insulin (l-4). Transfection of mutant complementary DNA coding for receptors lacking kinase activity results in increased binding but no increase in insulin action (2-4). Interestingly, mutations involving Tyr 960, a residue not believed to be phosphorylated in vivo, retain autophosphorylation and in vitro phosphotransferase activity but have decreased in vivo substrate phosphorylation and insulin action (5) including phosphatidylinositol 3-kinase activation (6). These results indicate that kinase activity is necessary but not sufficient for mediating insulin action. Although there is convincing evidence that autophosphorylation is essential for insulin action, no direct evidence indicates that tyrosine phosphorylation of subReceived September 11,199l. Address all correspondence and requests for reprints to: Dr. Robert A. Mooney, Department of Pathology and Laboratory Medicine, Universitv of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, New York 14642. *This work was supported by NIDDK Grant DK-38138 (to R.A.M.).
Materials
and Methods
Male Sprague-Dawley rats, weighing 125-150 g, were purchasedfrom CharlesRiver Breeding Laboratory (Wilmington, 1533
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Insulin Stimulates the Tyrosine Phosphorylation 6 1-Kilodalton Protein in Rat Adipocytes”
1534
AN INSULIN-DEPENDENT
61-kDa
Protein
ph43sphorylation
reaction conditions
Rat adipocyteswere suspendedat 2 x lo6 ceils/ml in phosphate-free Krebs-Ringer-HEPES (50 mM) containing 32Pi(300 pCi/0.5ml). After 2 hat 37 C, insulin wasaddedfor a subsequent P-min incubation unlessotherwise indicated. Cells were harvested by centrifuging through silicone oil and quick freezing in ethanol/dry ice. The cell layer was solubilized in 0.13 ml solubilizing buffer (1% SDS, 100 mM Tris at pH 7.4, 0.15 M NaCl), vortexed, and boiled 2 min. The solubilized cells were diluted lo-fold in 1% Triton X-100, 10 mg/ml deoxycholate, 0.15 M NaCl, and 100 mhi Tris at pH 7.4. After removal of the fat layer, the sampleswere mixed at 4 C overnight with antiphosphotyrosineantibody complexed to Protein G-Sepharose beads.Phosphotyrosine-containingproteins were releasedfrom the antibody complex with 20 mM p-nitrophenylphosphate, heatedin buffer containing 62 mM Tris (pH 6.8), 2% SDS, 10% glycerol, and 100mM dithiothreitol. Proteins were resolvedby electrophoresis on one-dimensional 5-15% SDS-polyacrylamide slab gelsusing the method of Laemmli (18). Autoradiography was performed at -70 C using preflashed film as describedby Laskey and Mills (19). A Helena Laboratories (Beaumont,TX) EDC computerized densitometerwas usedto quantitate autoradiographicresults. Phospbamino
acid analysis
Labeling of the insulin receptor and substrateswas as described above except that the amount of [32P]orthophosphate was increasedto 1.0 mCi/0.5 ml cell suspension.Immunoprecipitates from three sampleswere pooled for electrophoresis. The phosphoproteinbands were located in the dried gels by autoradiography and excised. The gel pieceswere washedseveral times in 50% methanol, dried, minced, and subjectedto direct acid hydrolysis (6~ HCl, 1.5 h at 110C). Phosphoamino acidswerepurified on Dowex 50-X8( H’) (20) and separatedby TLC at pH 3.5. One-dimen&nal
peptide
mapping
techniques
Phosphoproteinbands from dried (but not acid-fixed) gels were excised, rehydrated, and applied to the wells of a 15% SDS-polyacrylamide gel electrophoresis(PAGE) gel according to the procedureof Cleveland et al. (21). Staphylococcus aureus V8 proteaseor chymotrypsin were overlayed on the gel pieces and electrophoresedinto the stacking gel. After a 15-min in-
Endo.
PROTEIN
1992
cubation, the sampleswere electrophoretically separated,the gelswere fixed, dried, and the resulting phosphopeptidesvisualized by autoradiography. Cell fractionation
The frozen cell layers, labeledas above, were transferred to 0.2 ml homogenization buffer containing 10 mM Tris, pH 7.4, 0.25 M sucrose,10 mM NaF, 1 mM vanadate, 1 mM phenylmethanesulphonyl fluoride, 1 mg/ml benzamidine,30 mM sodium phosphate, 100,000U aprotinin, 1 mg/ml n-ethylmaleimide, 1 mg/ml bacitracin, and 10 mM @glycerophosphate.EDTA at 5 mM was added where indicated. Cells were homogenizedand then centrifuged at 1,000 X g for 1 min at 4 C in a Beckman microfuge (Beckman Instruments, Palo Alto, CA). The pellet and infranate were recovered,and the latter wascentrifuged at 10,000 X g for 15 min. The pellet and infranate were again recovered.The latter was centrifuged at 100,060x g (Beckman 42.2Ti fixed angle rotor) for 30 min. Pellets from the three centrifugations were solubilized in 0.13 ml solubilizing buffer (1% SDS, 100 mM Tris at pH 7.4,0.15 M NaCl), vortexed, and boiled 2 min. The 100,000x g supernatant wascombinedwith 0.13 ml 2~ solubilization buffer and processedas above.
Results
and Discussion
After a 2-min exposure to insulin (low7 M), three prominent, insulin-responsive, phosphorylated proteins
were immunoprecipitated from a suspension of prelabeled rat adipocytes using an antiphosphotyrosine antibody. The three proteins of 95 kDa, 160 kDa, and 61 kDa corresponded to the P-subunit of the insulin receptor (95 kDa) and two putative receptor substrates. A high molecular mass substrate has been observed in most insulinresponsive tissues (7-12). A 61 kDa protein was previously described in rat adipocytes by us and others using immunoblotting techniques (12, 14, 15). An additional diffuse, insulin-responsive band of approximately 80 kDa was frequently observed in these experiments. No effort has been made to characterize this protein band. Phosphorylation
of the 61-kDa protein demonstrated
essentially identical insulin concentration dependency to the autophosphorylated receptor (Fig. 1). Low levels of phosphorylation were observed at lo-’ M insulin with maximum effects seen at 10e7 M.
The 166kDa
high
molecular mass substrate was somewhat more sensitive, though less reproducible, with phosphorylation detectable at lo-“’ M insulin (Fig. 1B). The 61-kDa protein was maximally phosphorylated at 30 set as was the receptor and the 160-kDa phosphoprotein. No detectable change in level of phosphorylation
of any of the three proteins
was observed between 30 set and 10 min (data not shown). Labeling with [32P]orthophosphate provided information about the 61-kDa insulin-responsive protein which could not be obtained using immunoblots. This approach permitted a direct analysis and comparison of the phos-
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MA). Collagenase (Type I) was from Worthington (Freehold, NJ), and [32P]orthophosphate was from New England Nuclear (Boston, MA). Insulin was a generous gift from Lilly Laboratories (Indianapolis, IN). BSA (Cohn Fraction V) was from Sigma (St. Louis, MO). Protein G-Sepharose was from Pharmacia LKB Biotechnology (Piscataway, NJ), and cellulose TLC plates were from Analtech (Newark, DE). Prestained molecular mass markers for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were from Bethesda Research Laboratories (Bethesda, MD). A goat antiphosphotyrosine antibody was prepared (in collaboration with Dr. Morris White, Joslin Diabetes Institute (Boston MA) and Dr. James Livingston, University of Rochester) using phosphotyramine conjugated to keyhole limpet hemocyanin as the immunogen.
PHOSPHOTYROSYL
AN INSULIN-DEPENDENT
61-kDa
Molecular mass (kDa) ZOO-
-9
-8
-7
160 kDa
.-
95 kDa
c
61 kDa
4
LOG [INSULIN]
A
1.2 61 kDa
1
.._
0.0 -,,
-10
-0
.a
-7
-6
-5
LOG [INSULIN]
95kDa
1.0
LOG [INSULIN] 2.0
16OkDa
PROTEIN
1535
phoamino acids recovered from the 61-kDa protein, the insulin receptor, and the 160-kDa protein. Phosphoamino acid analysis revealed that the 61-kDa phosphoprotein contained relatively equal proportions of phosphotyrosine and phosphoserine under these experimental conditions (Fig. 2). No phosphothreonine was detected. In contrast, label from the 160-kDa protein was recovered primarily on phosphoserine. Since the proteins were immunoprecipitated with an antiphosphotyrosine antibody, this result would suggest that the 160-kDa protein has multiple phosphoserine sites per molecule. Interestingly, recent cloning and characterization of the high molecular mass receptor substrate, ~~185, (renamed IRS-l) (22) also revealed the predominance of phosphoserine over phosphotyrosine after insulin treatment. Finally, the insulin receptor contained both phosphoserine and phosphotyrosine with a majority of the label recovered on tyrosine residues. Little is known about the 61-kDa phosphoprotein. One possibility that had not been addressed in earlier reports was that this smaller phosphoprotein might be structurally related to the receptor or the 160-kDa protein, either physiologically or as an artifactual degradation product. Our results argue against these possibilities. The rapid solubilization of labeled adipocytes with boiling SDS decreased the possibility of proteolytic production of a 61-kDa fragment derived from the higher molecular mass phosphotyrosine-containing proteins. Cleveland peptide maps were prepared from the 61-kDa protein, the insulin receptor, and the 160-kDa protein to look for any sequence similarities. One dimensional peptide maps using Staph. aureus V8 proteinase revealed distinct peptide patterns for the three proteins (Fig. 3A). These maps were very reproducible with carryover of protease into the control lanes being the only inconsistency. Peptide maps with chymotrypsin also supported the observation
1.5
1.0
0.5
0.0 ~ -11
I3
-10
-0
4
-7
-6
4-p
-Ser
4-p
-Thr
C-P
-Tyr
-5
LOG [INSULIN]
1. Concentration-dependent effect of insulin on tyrosine-phosphorylated proteins in rat adipocytes. Rat adipocytes were prelabeled for 2 h with 0.3 mCi [32P]orthophosphate per cell suspension (1 x lo6 cells/O.5 ml). Suspensions were subsequently exposed to insulin for 2 min followed by rapid recovery of the cells, solubilization, and immunoprecipitation with an antiphosphotyrosine antibody. Proteins were separated by SDS-PAGE and visualized by autoradiography. A, Exposure time for autoradiograph was 48 h. B, Densitometric analysis of three experiments with results (mean If: SD) normalized to 3ZP incorporation at 10m6M insulin. Data point for 160 kDa at lo-’ M insulin represents an n = 1. FIG.
61 kDa
95 kDa
160
kDa
FIG. 2. Phosphoamino analysis of the insulin receptor and its putative substrates of 61 kDa and 160 kDa. Bat adipocytes were prelabeled for 2 h with 1.0 mCi [3ZP]orthophosphate per cell suspension (1 x lo6 cells/ 0.5 ml). Suspensions were subsequently exposed to insulin for 2 min followed by immunoprecipitation as described in Fig. 1. Phosphoprotein bands were excised, subjected to direct acid hydrolysis, and phosphoamino acids separated by electrophoresis as described in Materials and Methods.
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-10
.-
PHOSPHOTYROSYL
AN INSULIN-DEPENDENT
1536
61-kDa
w Molecular mass
PHOSPHOTYROSYL
Endo. Voll30.
PROTEIN
1992 No 3
123456769 Molecular
mass
(kDa) zoo-
(kDa)
97 -
hl60
+61
16-
95 kDa
kDa
43-
III
160kDa
Molec”l(aq! 2
mass
1
(kDa)
95kDa
61kDa
3
29 -
19 14
-
FIG. 3. One-dimensional peptide maps of the insulin receptor and its putative substrates of 61 kDa and 160 kDa. The 95kDa, Bl-kDa, and 160-kDa phosphoprotein bands, prepared as described in Fig. 2, were excised and subjected to Cleveland mapping on a 15% gel in the presence of SDS followed by autoradiographic visualization. a, Staph. aureus V8 proteinase per lane was 0 pg (lanes 1, 4, and 7), 0.05 rg (lanes 2,5, and 8), and 0.5 pg (lanes 3,6, and 9). b, Chymotrypsin per lane was 0.02 pg. A band at 190 kDa in the insulin receptor @-subunit lane (b, lane 2) is consistent with a 6-8 dimer.
that the 61-kDa protein was distinct from the receptor and the 160-kDa putative substrate (Fig. 3B). A simple subcellular fractionation by sequential centrifugation was performed to further characterize the 61kDa protein and its relationship to the insulin receptor and the 160-kDa protein (Fig. 4). Due to the small amount of labeled material and the lability of the phosphotyrosyl proteins, an extensive fractionation was not possible, and definitive cellular localization could not be accomplished. The insulin receptor was localized to the 1,000 X g and 10,000 X g pellets which contain the plasma membrane. The 160-kDa putative substrate was localized in equal proportions to the 100,000 X g pellet and super-
FIG. 4. Subcellular localization of the insulin receptor and its putative substrates of 61 kDa and 160 kDa. Rat adipocyte suspensions were prelabeled with [3ZP]orthophosphate and treated with insulin for 2 min. Harvested cells were homogenized in the presence of proteinase and phosphatase inhibitors and fractionated by sequential centrifugation as described in Materials and Methods. Lane 1, unfractionated, lanes 2 and 3, 1,000 X g pellet; lanes 4 and 5, 10,000 X g pellet; lanes 6 and 7, 100,000 X g pellet; lanes 8 and 9, 100,000 X g supernatant. Lanes 3,5, 7, and 9 contained 5 mM EDTA in the homogenization buffer.
natant. EDTA had no effect on this distribution. This is in contrast to the observations of Madoff et al. (23), in which membrane association of a 160-kDa insulin-dependent phosphotyrosyl protein in 3T3 Ll adipocytes required a divalent metal cation. The 61-kDa protein was likewise found primarily in the 100,000 x g pellet and supernatant but was also detected in the 1,000 x g pellet and to a lesser degree in the 10,000 X g pellet. Again, the inclusion of EDTA in the fractionation buffer had no appreciable effect on the results. The detection of the 61-kDa phosphoprotein in all fractions, some deficient in phosphorylated insulin receptor and others deficient in the 160-kDa protein, is additional evidence for the distinct character of this protein. To date, identification of substrates for the insulin receptor kinase has been difficult. Microtubule-associated protein kinase (24) and phosphatidylinositol 3-kinase (2526) are possible substrates. The most consistent response to insulin, however, in various tissues from many species has been the appearance of an insulindependent phosphotyrosyl protein of 160-185 kDa. It remains to be determined whether this protein is the same in all tissues and is the putative substrate, ~~185, first described by White et al. (7, 8) which is now cloned and sequenced (22). Although use of HzOz and/or vanadate has resulted in the generation of several more insulin-dependent phosphoproteins (27), their identity and physiological relevance remains uncertain. The insulindependent phosphotyrosyl protein of 60-65 kDa, though consistently observed in rat adipocytes (12-15), had not been characterized beyond its separation by SDS-PAGE. Based on these limited investigations of the 60-65 kDa
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+
kDa
AN INSULIN-DEPENDENT
61-kDa PHOSPHOTYROSYL
References 1. Ebina Y, Edery M, Ellis L, Standring D, Beaudoin J, Roth R, Rutter W 1985 Expression of a functional human insulin receptor from a cloned cDNA in Chinese hamster ovary cells. Proc Nat1
Acad Sci USA 82:8014-8018 2. Ebina Y, Araki E, Taira M, Shimada F, Mori M, Craik CS, Siddle K. Pierce SB. Roth RA. Rutter WJ 1987 Renlacement of lvsine residue 1030 in the putative ATP-binding region of the insulin receptor abolishes insulin- and antibody-stimulated glucose uptake and recentor kinase activitv. Proc Nat1 Acad Sci USA 84:704-708 3. Chou CK, Dull TJ, Russell-D& Gherzi R, Lebwohl AU, Rosen OM 1987 Human insulin receptors mutated at the ATP-binding site lack protein tyrosine kinase activity and fail to mediate postreceptor effects of insulin. J Biol Chem 2621842-1847 4. McClain DA, Maegawa H, Lee J, Dull TJ, Ullrich A, Olefsky JM 1987 A mutant insulin receptor with defective tyrosine kinase displays no biologic activity and does not undergo endocytosis. J Biol Chem 262:14663-14671 5. White MF, Livingston JN, Backer JM, Lauris V, Dull TJ, Ullrich A, Kahn CR 1988 Mutation of the insulin receptor at tyrosine 960 inhibits signal transmission but does not affect its tyrosine kinase activity. Cell 54:641-649 6. Kapeller R, Chen KS, Yoakim M, Schaffhausen BS, Backer J, White MF, Cantley LC, Ruderman NB 1991 Mutations in the juxtamembrane region of the insulin receptor impair activation of nhosnhatidvlinositol 3-kinase bv insulin. Mol Endocrinol 5:769777 7. White MF, Maron R, Kahn CR 1985 Insulin rapidly stimulates tvrosine uhosnhorvlation of a Mr-185,000 nrotein in intact cells. _ Nature 318183-186 8. White MF, Stegmann EW, Dull TJ, Ullrich A, Kahn CR 1987 Characterization of an endogenous substrate of the insulin receptor in cultured cells. J Biol Chem 262:9769-9777 9. Gibbs EM, Allard WJ, Lienhard GE 1986 The glucose transporter in 3T3-Ll adipocytes is phosphorylated in response to phorbol ester but not in response to insulin. J Biol Chem 261:16597-16601 10. Machicao F, Haring H, White MF, Carrascosa JM, Obermaier B, Wieland OH 1987 An Mr 180.000 nrotein is an endozenous substrate for the insulin-receptor-associated tyrosine kinaie in human placenta. Biochem J 243:797-801 11. Shemer J, Adamo M, Wilson GL, Heffez D, Zick Y, LeRoith D 1987 Insulin and insulin-like growth factor-I stimulate a common endogenous phosphoprotein substrate (pp 185) in intact neuroblastoma cells. J Biol Chem 262:15476-15482 12. Mooney RA, Bordwell KL, Luhowskyj S, Casnellie JE 1989 The insulin-like effect of vanadate on lipolysis in rat adipocytes is not accompanied by an insulin-like effect on tyrosine phosphorylation. Endocrinology 124422-429 13. Haring HU, White MF, Machicao F, Ermel B, Schleicher E, Obermaier B 1987 Insulin rapidly stimulates phosphorylation of a 46-kDa membrane protein on tyrosine residues as well as phosphorylation of several soluble proteins in intact fat cells. Proc Nat1 Acad Sci USA 84:113-117 14. Momoura K, Tobe K, Seyama Y, Takaku F, Kasuga M 1988 Insulin-induced tyrosine-phosphorylation in intact rat adipocytes. Biochem Biophys Res Commun 155:1181-1186 15. Ezaki 0 1989 IIb group metal ions (Zn*+, Cd*+, He) stimulate glucose transport activity by post-insulin receptor kinase mechanism in rat adipocytes. J Biol Chem 264161%16122 16. Colca JR, DeWald DB, Pearson JD, Palazuk BJ, Laurino JP, McDonald JM 1987 Insulin stimulates the phosphorylation of calmodulin in intact adipocytes. J Biol Chem 262:11399-11402 17. Bernier M, Laird DM, Lane MD 1987 Insulin-activated tyrosine phosphorylation of a 15-kilodalton protein in intact 3T3-Ll adipocytes. Proc Nat1 Acad Sci USA 84:1844-1848 18. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 19. Laskey RA, Mills AD 1977 Enhanced autoradiographic detection of 32P and ‘%I using intensifying screens and hypersensitized film. FEBS Lett 82:314-316 20. Martensen TM 1984 Chemical properties, isolation, and analysis of o-phosphates in proteins. Methods Enzymol 107:3-23 21. Cleveland DW, Fischer SG, Kirschner MW, Laemmli UK 1977 Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analvsis bv eel electronhosesis. J Biol Chem 252:1102-1106 22. Sun XJ,“Rothkiberg P, Kahn CR, Backer JM, Araki E, Wilden
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protein, it was not possible to conclude that the low molecular mass phosphotyrosyl protein was not derived from or related to the insulin receptor or the high molecular mass receptor substrate. Our data provide evidence that the 61-kDa insulin-dependent phosphoprotein is distinct from these proteins. Additionally, the time course of its phosphorylation and its insulin dependency provide an argument that this protein should be included in the short list of putative substrates for the insulin receptor tyrosine kinase. As with the 160-kDa protein, no functional role for the 61-kDa protein is yet known. Like the 160-kDa protein and the insulin receptor, the 61-kDa protein is both serine and tyrosine phosphorylated. This is a frequent observation with tyrosine phosphorylated proteins and suggests that the cellular role of the 61-kDa protein requires multiple phosphorylation signals mediated by both serine and tyrosine kinases. An interesting characteristic of this protein is its apparent ubiquitous distribution within the cell. It was found in both the low and high speed pellets as well as the cytosol. This may indicate that the 61-kDa protein actually represents a closely related family of proteins or a protein which can associate with multiple elements within the cell. O’Brien et al. (28) have described a putative insulin receptor tyrosine kinase substrate of 60 kDa in the NIH 3T3 HIR3.5 cell line. The protein bound to GDP-agarose, and from this, the authors suggested that the protein may be a novel GTP-binding protein. Based upon our cell fractionation data, however, the 61-kDa phosphotyrosyl-protein described in this report would not be a classic plasma membrane-associated G protein. The majority of the 61-kDa protein is not found in the plasma membrane but in the postplasma membrane microsomal fraction and cytosol. Secondly, the 61-kDa protein has the incorrect size for a classic G protein which has a subunit molecular mass of 40-45 kDa for a-subunits, 37 kDa for B-subunits, and 8-10 kDa for y-subunits (29). In summary, a unique 61-kDa tyrosine-phosphorylated protein has been described in insulin-stimulated rat adipocytes. It is very rapidly phosphorylated in response to insulin and demonstrates a very similar hormone concentration dependence to the insulin receptor and a high molecular mass receptor substrate of 160 kDa. This 61kDa protein appears to be both a putative substrate for the insulin receptor tyrosine kinase and a substrate for a serine kinase. Identification of this protein may further our understanding of insulin signal transduction and insulin action.
PROTEIN
1538
AN
INSULIN-DEPENDENT
61-kDa
PROTEIN
Endo. Voll30.
1992 No 3
26. Endemann G, Yonezawa K, Roth RA 1990 Phosphatidylinositol kinase or an associated protein is a substrate for the insulin receutor tyrosine kinase. J Biol Chem 265:396-400 27. Heffetx D; Zick Y 1989 H201 potentiates phosphorylation of novel putative substrates for the insulin receptor kinase in intact Fao cells. J Biol Chem 2641012610132 28. O’Brien R, Houslay MD, Brindle NP, Milligan G, Whittaker J, Siddle K 1989 Binding to GDP-agarose identifies a novel 60 kDa substrate for the insulin receptor tyrosyl kinase in mouse NIH3T3 cells expressing high concentrations of the human insulin receptor. Biochem Biophys Res Commun 158743-748 29. Casey PJ, Gilman AG 1988 G protein involvement in receptoreffector coupling. J Biol Chem 263:2577-2580
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PA, Cahill DA, Goldstein BJ, White MF 1991 Structure of the insulin receptor substrate IRS-l defines a unique signal transduction protein. Nature 352:73-II 23. Madoff DH, Martensen TM, Lane MD 1988 Insulin and insulinlike growth factor 1 stimulate the phosphorylation on tyrosine of a 160 kDa cytosolic protein in 3T3-Ll adipocytes. Biochem J 252:715 24. Ray LB, Sturgill TW 1988 Insulin-stimulated microtubule-associated protein kinase is phosphorylated on tyrosine and threonine in uiuo. Proc Nat1 Acad Sci USA 853753-3757 25. Ruderman NB, Kapeller R, White MF, Cantley LC 1990 Activation of phosphatidylinositol 3-kinase by insulin. Proc Nat1 Acad Sci USA 87:1411-1415
PHOSPHOTYROSYL
Vol. 130, No. 3 Printed in U.S.A.
Society
ROBERT
A. MOONEY
AND KATHY
Department of Pathology and Laboratory Dentistty, Rochester, New York 14642
of a
L. BORDWELL
Medicine,
University
of
Rochester School of Medicine
ABSTRACT. Insulin stimulated the tyrosine phosphorylation of a 61-kilodalton &Da) protein in rat adipocytes prelabeled for 2 h with 13*Plorthouhosnhate. Tvrosine phosphorvlation of this 61-kDa &o&in diipla$d very- similar- in&in concentration dependency to receptor autophosphorylation and tyrosine phosphorylation of a high molecular mass receptor substrata of 160 kDa. Phoaphorylation of the Bl-kDa protein was very rapid with maximum labeling attained at 30 set, paralleling that of the other two proteins. Phosphoamino acid analysis revealed that each of the insulin-responsive phosphoproteins contained phosphoserine as well as phosphotyrosine, though the ratio of two phosphoamino acids recovered from each protein differed. The 61-kDa protein yielded relatively equal proportions of phosphoserine and phosphotyrosine. In contrast, the insulin receptor yielded relatively more label on phosphotyrosine than phosphoserine, whereas label incorporated into the 160-kDa protein was
and
recovered primarily on phosphoserine. Cleveland peptide maps using either Staphylococcus aurew V8 proteinase or chymotrypsin revealed no similarities between the 61-kDa protein and the other tyrosine phosphorylated proteins. With subcellular fractionation, the 160-kDa protein was found in equal proportions in the high speed pellet (100,000 g) and supernatant. The 61kDa protein had a similar distribution to that of the 160-kDa protein but was also detected in the low speed pallet (10,000 g). The insulin receptor was localized to the low speed pellet. In summary, rat adipocytes contain an insulin-dependent phosphotyrosyl protein of 61 kDa which is distinct from the more prominent high molecular mass receptor substrate. Thii 61-kDa protein has characteristics consistent with it being a substrate for the insulin receptor tyrosine kinase. (Endocrinology 130: 1533-1538,1992)
S
strates is also essential. This may be partly due to the difficulty in identifying and studying these rare phosphorylation events. Nonetheless, several potential substrates that are phosphorylated on tyrosine residues in an insulin-dependent manner have now been recognized. White et al. (7,8) described a 185K phosphoprotein from the Fao hepatoma cell line using an antiphosphotyrosine antibody. Insulin-dependent phosphotyrosyl proteins of similar molecular mass have also been observed in other insulin-responsive tissues (g-11), including rat adipocytes (12). We and others have observed additional insulin-dependent phosphotyrosine-containing proteins in rat adipocytes including those of 60-65 kDa (12-l@, 46 kDa (13), 17 kDa (calmodulin) (16), and 15 kDa (17). The 60-65 kDa (61-kDa in our studies) insulin-dependent phosphotyrosine-containing protein is of particular interest because it has consistently been observed in rat adipocytes. The very limited knowledge of the 60-65 kDa phosphotyrosine-containing protein in rat adipocytes, however, has been obtained through immunoblotting techniques. Direct characterization of the phosphoprotein has not been reported and is the objective of this study.
UBSTANTIAL evidence links insulin receptor tyrosine kinase activity to insulin signal transduction and insulin action. Most convincing of this evidence comes from the molecular biological approach. Cell lines transfected with human insulin receptor complementary DNA show increased insulin binding and increased sensitivity to insulin (l-4). Transfection of mutant complementary DNA coding for receptors lacking kinase activity results in increased binding but no increase in insulin action (2-4). Interestingly, mutations involving Tyr 960, a residue not believed to be phosphorylated in vivo, retain autophosphorylation and in vitro phosphotransferase activity but have decreased in vivo substrate phosphorylation and insulin action (5) including phosphatidylinositol 3-kinase activation (6). These results indicate that kinase activity is necessary but not sufficient for mediating insulin action. Although there is convincing evidence that autophosphorylation is essential for insulin action, no direct evidence indicates that tyrosine phosphorylation of subReceived September 11,199l. Address all correspondence and requests for reprints to: Dr. Robert A. Mooney, Department of Pathology and Laboratory Medicine, Universitv of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, New York 14642. *This work was supported by NIDDK Grant DK-38138 (to R.A.M.).
Materials
and Methods
Male Sprague-Dawley rats, weighing 125-150 g, were purchasedfrom CharlesRiver Breeding Laboratory (Wilmington, 1533
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Insulin Stimulates the Tyrosine Phosphorylation 6 1-Kilodalton Protein in Rat Adipocytes”
1534
AN INSULIN-DEPENDENT
61-kDa
Protein
ph43sphorylation
reaction conditions
Rat adipocyteswere suspendedat 2 x lo6 ceils/ml in phosphate-free Krebs-Ringer-HEPES (50 mM) containing 32Pi(300 pCi/0.5ml). After 2 hat 37 C, insulin wasaddedfor a subsequent P-min incubation unlessotherwise indicated. Cells were harvested by centrifuging through silicone oil and quick freezing in ethanol/dry ice. The cell layer was solubilized in 0.13 ml solubilizing buffer (1% SDS, 100 mM Tris at pH 7.4, 0.15 M NaCl), vortexed, and boiled 2 min. The solubilized cells were diluted lo-fold in 1% Triton X-100, 10 mg/ml deoxycholate, 0.15 M NaCl, and 100 mhi Tris at pH 7.4. After removal of the fat layer, the sampleswere mixed at 4 C overnight with antiphosphotyrosineantibody complexed to Protein G-Sepharose beads.Phosphotyrosine-containingproteins were releasedfrom the antibody complex with 20 mM p-nitrophenylphosphate, heatedin buffer containing 62 mM Tris (pH 6.8), 2% SDS, 10% glycerol, and 100mM dithiothreitol. Proteins were resolvedby electrophoresis on one-dimensional 5-15% SDS-polyacrylamide slab gelsusing the method of Laemmli (18). Autoradiography was performed at -70 C using preflashed film as describedby Laskey and Mills (19). A Helena Laboratories (Beaumont,TX) EDC computerized densitometerwas usedto quantitate autoradiographicresults. Phospbamino
acid analysis
Labeling of the insulin receptor and substrateswas as described above except that the amount of [32P]orthophosphate was increasedto 1.0 mCi/0.5 ml cell suspension.Immunoprecipitates from three sampleswere pooled for electrophoresis. The phosphoproteinbands were located in the dried gels by autoradiography and excised. The gel pieceswere washedseveral times in 50% methanol, dried, minced, and subjectedto direct acid hydrolysis (6~ HCl, 1.5 h at 110C). Phosphoamino acidswerepurified on Dowex 50-X8( H’) (20) and separatedby TLC at pH 3.5. One-dimen&nal
peptide
mapping
techniques
Phosphoproteinbands from dried (but not acid-fixed) gels were excised, rehydrated, and applied to the wells of a 15% SDS-polyacrylamide gel electrophoresis(PAGE) gel according to the procedureof Cleveland et al. (21). Staphylococcus aureus V8 proteaseor chymotrypsin were overlayed on the gel pieces and electrophoresedinto the stacking gel. After a 15-min in-
Endo.
PROTEIN
1992
cubation, the sampleswere electrophoretically separated,the gelswere fixed, dried, and the resulting phosphopeptidesvisualized by autoradiography. Cell fractionation
The frozen cell layers, labeledas above, were transferred to 0.2 ml homogenization buffer containing 10 mM Tris, pH 7.4, 0.25 M sucrose,10 mM NaF, 1 mM vanadate, 1 mM phenylmethanesulphonyl fluoride, 1 mg/ml benzamidine,30 mM sodium phosphate, 100,000U aprotinin, 1 mg/ml n-ethylmaleimide, 1 mg/ml bacitracin, and 10 mM @glycerophosphate.EDTA at 5 mM was added where indicated. Cells were homogenizedand then centrifuged at 1,000 X g for 1 min at 4 C in a Beckman microfuge (Beckman Instruments, Palo Alto, CA). The pellet and infranate were recovered,and the latter wascentrifuged at 10,000 X g for 15 min. The pellet and infranate were again recovered.The latter was centrifuged at 100,060x g (Beckman 42.2Ti fixed angle rotor) for 30 min. Pellets from the three centrifugations were solubilized in 0.13 ml solubilizing buffer (1% SDS, 100 mM Tris at pH 7.4,0.15 M NaCl), vortexed, and boiled 2 min. The 100,000x g supernatant wascombinedwith 0.13 ml 2~ solubilization buffer and processedas above.
Results
and Discussion
After a 2-min exposure to insulin (low7 M), three prominent, insulin-responsive, phosphorylated proteins
were immunoprecipitated from a suspension of prelabeled rat adipocytes using an antiphosphotyrosine antibody. The three proteins of 95 kDa, 160 kDa, and 61 kDa corresponded to the P-subunit of the insulin receptor (95 kDa) and two putative receptor substrates. A high molecular mass substrate has been observed in most insulinresponsive tissues (7-12). A 61 kDa protein was previously described in rat adipocytes by us and others using immunoblotting techniques (12, 14, 15). An additional diffuse, insulin-responsive band of approximately 80 kDa was frequently observed in these experiments. No effort has been made to characterize this protein band. Phosphorylation
of the 61-kDa protein demonstrated
essentially identical insulin concentration dependency to the autophosphorylated receptor (Fig. 1). Low levels of phosphorylation were observed at lo-’ M insulin with maximum effects seen at 10e7 M.
The 166kDa
high
molecular mass substrate was somewhat more sensitive, though less reproducible, with phosphorylation detectable at lo-“’ M insulin (Fig. 1B). The 61-kDa protein was maximally phosphorylated at 30 set as was the receptor and the 160-kDa phosphoprotein. No detectable change in level of phosphorylation
of any of the three proteins
was observed between 30 set and 10 min (data not shown). Labeling with [32P]orthophosphate provided information about the 61-kDa insulin-responsive protein which could not be obtained using immunoblots. This approach permitted a direct analysis and comparison of the phos-
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MA). Collagenase (Type I) was from Worthington (Freehold, NJ), and [32P]orthophosphate was from New England Nuclear (Boston, MA). Insulin was a generous gift from Lilly Laboratories (Indianapolis, IN). BSA (Cohn Fraction V) was from Sigma (St. Louis, MO). Protein G-Sepharose was from Pharmacia LKB Biotechnology (Piscataway, NJ), and cellulose TLC plates were from Analtech (Newark, DE). Prestained molecular mass markers for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were from Bethesda Research Laboratories (Bethesda, MD). A goat antiphosphotyrosine antibody was prepared (in collaboration with Dr. Morris White, Joslin Diabetes Institute (Boston MA) and Dr. James Livingston, University of Rochester) using phosphotyramine conjugated to keyhole limpet hemocyanin as the immunogen.
PHOSPHOTYROSYL
AN INSULIN-DEPENDENT
61-kDa
Molecular mass (kDa) ZOO-
-9
-8
-7
160 kDa
.-
95 kDa
c
61 kDa
4
LOG [INSULIN]
A
1.2 61 kDa
1
.._
0.0 -,,
-10
-0
.a
-7
-6
-5
LOG [INSULIN]
95kDa
1.0
LOG [INSULIN] 2.0
16OkDa
PROTEIN
1535
phoamino acids recovered from the 61-kDa protein, the insulin receptor, and the 160-kDa protein. Phosphoamino acid analysis revealed that the 61-kDa phosphoprotein contained relatively equal proportions of phosphotyrosine and phosphoserine under these experimental conditions (Fig. 2). No phosphothreonine was detected. In contrast, label from the 160-kDa protein was recovered primarily on phosphoserine. Since the proteins were immunoprecipitated with an antiphosphotyrosine antibody, this result would suggest that the 160-kDa protein has multiple phosphoserine sites per molecule. Interestingly, recent cloning and characterization of the high molecular mass receptor substrate, ~~185, (renamed IRS-l) (22) also revealed the predominance of phosphoserine over phosphotyrosine after insulin treatment. Finally, the insulin receptor contained both phosphoserine and phosphotyrosine with a majority of the label recovered on tyrosine residues. Little is known about the 61-kDa phosphoprotein. One possibility that had not been addressed in earlier reports was that this smaller phosphoprotein might be structurally related to the receptor or the 160-kDa protein, either physiologically or as an artifactual degradation product. Our results argue against these possibilities. The rapid solubilization of labeled adipocytes with boiling SDS decreased the possibility of proteolytic production of a 61-kDa fragment derived from the higher molecular mass phosphotyrosine-containing proteins. Cleveland peptide maps were prepared from the 61-kDa protein, the insulin receptor, and the 160-kDa protein to look for any sequence similarities. One dimensional peptide maps using Staph. aureus V8 proteinase revealed distinct peptide patterns for the three proteins (Fig. 3A). These maps were very reproducible with carryover of protease into the control lanes being the only inconsistency. Peptide maps with chymotrypsin also supported the observation
1.5
1.0
0.5
0.0 ~ -11
I3
-10
-0
4
-7
-6
4-p
-Ser
4-p
-Thr
C-P
-Tyr
-5
LOG [INSULIN]
1. Concentration-dependent effect of insulin on tyrosine-phosphorylated proteins in rat adipocytes. Rat adipocytes were prelabeled for 2 h with 0.3 mCi [32P]orthophosphate per cell suspension (1 x lo6 cells/O.5 ml). Suspensions were subsequently exposed to insulin for 2 min followed by rapid recovery of the cells, solubilization, and immunoprecipitation with an antiphosphotyrosine antibody. Proteins were separated by SDS-PAGE and visualized by autoradiography. A, Exposure time for autoradiograph was 48 h. B, Densitometric analysis of three experiments with results (mean If: SD) normalized to 3ZP incorporation at 10m6M insulin. Data point for 160 kDa at lo-’ M insulin represents an n = 1. FIG.
61 kDa
95 kDa
160
kDa
FIG. 2. Phosphoamino analysis of the insulin receptor and its putative substrates of 61 kDa and 160 kDa. Bat adipocytes were prelabeled for 2 h with 1.0 mCi [3ZP]orthophosphate per cell suspension (1 x lo6 cells/ 0.5 ml). Suspensions were subsequently exposed to insulin for 2 min followed by immunoprecipitation as described in Fig. 1. Phosphoprotein bands were excised, subjected to direct acid hydrolysis, and phosphoamino acids separated by electrophoresis as described in Materials and Methods.
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-10
.-
PHOSPHOTYROSYL
AN INSULIN-DEPENDENT
1536
61-kDa
w Molecular mass
PHOSPHOTYROSYL
Endo. Voll30.
PROTEIN
1992 No 3
123456769 Molecular
mass
(kDa) zoo-
(kDa)
97 -
hl60
+61
16-
95 kDa
kDa
43-
III
160kDa
Molec”l(aq! 2
mass
1
(kDa)
95kDa
61kDa
3
29 -
19 14
-
FIG. 3. One-dimensional peptide maps of the insulin receptor and its putative substrates of 61 kDa and 160 kDa. The 95kDa, Bl-kDa, and 160-kDa phosphoprotein bands, prepared as described in Fig. 2, were excised and subjected to Cleveland mapping on a 15% gel in the presence of SDS followed by autoradiographic visualization. a, Staph. aureus V8 proteinase per lane was 0 pg (lanes 1, 4, and 7), 0.05 rg (lanes 2,5, and 8), and 0.5 pg (lanes 3,6, and 9). b, Chymotrypsin per lane was 0.02 pg. A band at 190 kDa in the insulin receptor @-subunit lane (b, lane 2) is consistent with a 6-8 dimer.
that the 61-kDa protein was distinct from the receptor and the 160-kDa putative substrate (Fig. 3B). A simple subcellular fractionation by sequential centrifugation was performed to further characterize the 61kDa protein and its relationship to the insulin receptor and the 160-kDa protein (Fig. 4). Due to the small amount of labeled material and the lability of the phosphotyrosyl proteins, an extensive fractionation was not possible, and definitive cellular localization could not be accomplished. The insulin receptor was localized to the 1,000 X g and 10,000 X g pellets which contain the plasma membrane. The 160-kDa putative substrate was localized in equal proportions to the 100,000 X g pellet and super-
FIG. 4. Subcellular localization of the insulin receptor and its putative substrates of 61 kDa and 160 kDa. Rat adipocyte suspensions were prelabeled with [3ZP]orthophosphate and treated with insulin for 2 min. Harvested cells were homogenized in the presence of proteinase and phosphatase inhibitors and fractionated by sequential centrifugation as described in Materials and Methods. Lane 1, unfractionated, lanes 2 and 3, 1,000 X g pellet; lanes 4 and 5, 10,000 X g pellet; lanes 6 and 7, 100,000 X g pellet; lanes 8 and 9, 100,000 X g supernatant. Lanes 3,5, 7, and 9 contained 5 mM EDTA in the homogenization buffer.
natant. EDTA had no effect on this distribution. This is in contrast to the observations of Madoff et al. (23), in which membrane association of a 160-kDa insulin-dependent phosphotyrosyl protein in 3T3 Ll adipocytes required a divalent metal cation. The 61-kDa protein was likewise found primarily in the 100,000 x g pellet and supernatant but was also detected in the 1,000 x g pellet and to a lesser degree in the 10,000 X g pellet. Again, the inclusion of EDTA in the fractionation buffer had no appreciable effect on the results. The detection of the 61-kDa phosphoprotein in all fractions, some deficient in phosphorylated insulin receptor and others deficient in the 160-kDa protein, is additional evidence for the distinct character of this protein. To date, identification of substrates for the insulin receptor kinase has been difficult. Microtubule-associated protein kinase (24) and phosphatidylinositol 3-kinase (2526) are possible substrates. The most consistent response to insulin, however, in various tissues from many species has been the appearance of an insulindependent phosphotyrosyl protein of 160-185 kDa. It remains to be determined whether this protein is the same in all tissues and is the putative substrate, ~~185, first described by White et al. (7, 8) which is now cloned and sequenced (22). Although use of HzOz and/or vanadate has resulted in the generation of several more insulin-dependent phosphoproteins (27), their identity and physiological relevance remains uncertain. The insulindependent phosphotyrosyl protein of 60-65 kDa, though consistently observed in rat adipocytes (12-15), had not been characterized beyond its separation by SDS-PAGE. Based on these limited investigations of the 60-65 kDa
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+
kDa
AN INSULIN-DEPENDENT
61-kDa PHOSPHOTYROSYL
References 1. Ebina Y, Edery M, Ellis L, Standring D, Beaudoin J, Roth R, Rutter W 1985 Expression of a functional human insulin receptor from a cloned cDNA in Chinese hamster ovary cells. Proc Nat1
Acad Sci USA 82:8014-8018 2. Ebina Y, Araki E, Taira M, Shimada F, Mori M, Craik CS, Siddle K. Pierce SB. Roth RA. Rutter WJ 1987 Renlacement of lvsine residue 1030 in the putative ATP-binding region of the insulin receptor abolishes insulin- and antibody-stimulated glucose uptake and recentor kinase activitv. Proc Nat1 Acad Sci USA 84:704-708 3. Chou CK, Dull TJ, Russell-D& Gherzi R, Lebwohl AU, Rosen OM 1987 Human insulin receptors mutated at the ATP-binding site lack protein tyrosine kinase activity and fail to mediate postreceptor effects of insulin. J Biol Chem 2621842-1847 4. McClain DA, Maegawa H, Lee J, Dull TJ, Ullrich A, Olefsky JM 1987 A mutant insulin receptor with defective tyrosine kinase displays no biologic activity and does not undergo endocytosis. J Biol Chem 262:14663-14671 5. White MF, Livingston JN, Backer JM, Lauris V, Dull TJ, Ullrich A, Kahn CR 1988 Mutation of the insulin receptor at tyrosine 960 inhibits signal transmission but does not affect its tyrosine kinase activity. Cell 54:641-649 6. Kapeller R, Chen KS, Yoakim M, Schaffhausen BS, Backer J, White MF, Cantley LC, Ruderman NB 1991 Mutations in the juxtamembrane region of the insulin receptor impair activation of nhosnhatidvlinositol 3-kinase bv insulin. Mol Endocrinol 5:769777 7. White MF, Maron R, Kahn CR 1985 Insulin rapidly stimulates tvrosine uhosnhorvlation of a Mr-185,000 nrotein in intact cells. _ Nature 318183-186 8. White MF, Stegmann EW, Dull TJ, Ullrich A, Kahn CR 1987 Characterization of an endogenous substrate of the insulin receptor in cultured cells. J Biol Chem 262:9769-9777 9. Gibbs EM, Allard WJ, Lienhard GE 1986 The glucose transporter in 3T3-Ll adipocytes is phosphorylated in response to phorbol ester but not in response to insulin. J Biol Chem 261:16597-16601 10. Machicao F, Haring H, White MF, Carrascosa JM, Obermaier B, Wieland OH 1987 An Mr 180.000 nrotein is an endozenous substrate for the insulin-receptor-associated tyrosine kinaie in human placenta. Biochem J 243:797-801 11. Shemer J, Adamo M, Wilson GL, Heffez D, Zick Y, LeRoith D 1987 Insulin and insulin-like growth factor-I stimulate a common endogenous phosphoprotein substrate (pp 185) in intact neuroblastoma cells. J Biol Chem 262:15476-15482 12. Mooney RA, Bordwell KL, Luhowskyj S, Casnellie JE 1989 The insulin-like effect of vanadate on lipolysis in rat adipocytes is not accompanied by an insulin-like effect on tyrosine phosphorylation. Endocrinology 124422-429 13. Haring HU, White MF, Machicao F, Ermel B, Schleicher E, Obermaier B 1987 Insulin rapidly stimulates phosphorylation of a 46-kDa membrane protein on tyrosine residues as well as phosphorylation of several soluble proteins in intact fat cells. Proc Nat1 Acad Sci USA 84:113-117 14. Momoura K, Tobe K, Seyama Y, Takaku F, Kasuga M 1988 Insulin-induced tyrosine-phosphorylation in intact rat adipocytes. Biochem Biophys Res Commun 155:1181-1186 15. Ezaki 0 1989 IIb group metal ions (Zn*+, Cd*+, He) stimulate glucose transport activity by post-insulin receptor kinase mechanism in rat adipocytes. J Biol Chem 264161%16122 16. Colca JR, DeWald DB, Pearson JD, Palazuk BJ, Laurino JP, McDonald JM 1987 Insulin stimulates the phosphorylation of calmodulin in intact adipocytes. J Biol Chem 262:11399-11402 17. Bernier M, Laird DM, Lane MD 1987 Insulin-activated tyrosine phosphorylation of a 15-kilodalton protein in intact 3T3-Ll adipocytes. Proc Nat1 Acad Sci USA 84:1844-1848 18. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 19. Laskey RA, Mills AD 1977 Enhanced autoradiographic detection of 32P and ‘%I using intensifying screens and hypersensitized film. FEBS Lett 82:314-316 20. Martensen TM 1984 Chemical properties, isolation, and analysis of o-phosphates in proteins. Methods Enzymol 107:3-23 21. Cleveland DW, Fischer SG, Kirschner MW, Laemmli UK 1977 Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analvsis bv eel electronhosesis. J Biol Chem 252:1102-1106 22. Sun XJ,“Rothkiberg P, Kahn CR, Backer JM, Araki E, Wilden
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protein, it was not possible to conclude that the low molecular mass phosphotyrosyl protein was not derived from or related to the insulin receptor or the high molecular mass receptor substrate. Our data provide evidence that the 61-kDa insulin-dependent phosphoprotein is distinct from these proteins. Additionally, the time course of its phosphorylation and its insulin dependency provide an argument that this protein should be included in the short list of putative substrates for the insulin receptor tyrosine kinase. As with the 160-kDa protein, no functional role for the 61-kDa protein is yet known. Like the 160-kDa protein and the insulin receptor, the 61-kDa protein is both serine and tyrosine phosphorylated. This is a frequent observation with tyrosine phosphorylated proteins and suggests that the cellular role of the 61-kDa protein requires multiple phosphorylation signals mediated by both serine and tyrosine kinases. An interesting characteristic of this protein is its apparent ubiquitous distribution within the cell. It was found in both the low and high speed pellets as well as the cytosol. This may indicate that the 61-kDa protein actually represents a closely related family of proteins or a protein which can associate with multiple elements within the cell. O’Brien et al. (28) have described a putative insulin receptor tyrosine kinase substrate of 60 kDa in the NIH 3T3 HIR3.5 cell line. The protein bound to GDP-agarose, and from this, the authors suggested that the protein may be a novel GTP-binding protein. Based upon our cell fractionation data, however, the 61-kDa phosphotyrosyl-protein described in this report would not be a classic plasma membrane-associated G protein. The majority of the 61-kDa protein is not found in the plasma membrane but in the postplasma membrane microsomal fraction and cytosol. Secondly, the 61-kDa protein has the incorrect size for a classic G protein which has a subunit molecular mass of 40-45 kDa for a-subunits, 37 kDa for B-subunits, and 8-10 kDa for y-subunits (29). In summary, a unique 61-kDa tyrosine-phosphorylated protein has been described in insulin-stimulated rat adipocytes. It is very rapidly phosphorylated in response to insulin and demonstrates a very similar hormone concentration dependence to the insulin receptor and a high molecular mass receptor substrate of 160 kDa. This 61kDa protein appears to be both a putative substrate for the insulin receptor tyrosine kinase and a substrate for a serine kinase. Identification of this protein may further our understanding of insulin signal transduction and insulin action.
PROTEIN
1538
AN
INSULIN-DEPENDENT
61-kDa
PROTEIN
Endo. Voll30.
1992 No 3
26. Endemann G, Yonezawa K, Roth RA 1990 Phosphatidylinositol kinase or an associated protein is a substrate for the insulin receutor tyrosine kinase. J Biol Chem 265:396-400 27. Heffetx D; Zick Y 1989 H201 potentiates phosphorylation of novel putative substrates for the insulin receptor kinase in intact Fao cells. J Biol Chem 2641012610132 28. O’Brien R, Houslay MD, Brindle NP, Milligan G, Whittaker J, Siddle K 1989 Binding to GDP-agarose identifies a novel 60 kDa substrate for the insulin receptor tyrosyl kinase in mouse NIH3T3 cells expressing high concentrations of the human insulin receptor. Biochem Biophys Res Commun 158743-748 29. Casey PJ, Gilman AG 1988 G protein involvement in receptoreffector coupling. J Biol Chem 263:2577-2580
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PA, Cahill DA, Goldstein BJ, White MF 1991 Structure of the insulin receptor substrate IRS-l defines a unique signal transduction protein. Nature 352:73-II 23. Madoff DH, Martensen TM, Lane MD 1988 Insulin and insulinlike growth factor 1 stimulate the phosphorylation on tyrosine of a 160 kDa cytosolic protein in 3T3-Ll adipocytes. Biochem J 252:715 24. Ray LB, Sturgill TW 1988 Insulin-stimulated microtubule-associated protein kinase is phosphorylated on tyrosine and threonine in uiuo. Proc Nat1 Acad Sci USA 853753-3757 25. Ruderman NB, Kapeller R, White MF, Cantley LC 1990 Activation of phosphatidylinositol 3-kinase by insulin. Proc Nat1 Acad Sci USA 87:1411-1415
PHOSPHOTYROSYL
