©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a 190-kDa Protein as a Novel Substrate for the Insulin Receptor Kinase Functionally Similar to Insulin Receptor Substrate-1 (*)

(Received for publication, October 31, 1994; and in revised form, January 20, 1995)

Kazuyuki Tobe (1) Hiroyuki Tamemoto (1) Toshimasa Yamauchi (1) Shinichi Aizawa (2) Yoshio Yazaki (1) Takashi Kadowaki (1) (3)(§)

From the  (1)Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan, the (2)Laboratory of Morphogenesis, IMEG, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860, Japan, and the (3)Institute for Diabetes Care and Research, Asahi Life Foundation, Marunouchi, Chiyoda-ku, Tokyo 100, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Recently, we generated mice with a targeted disruption of the insulin receptor substrate-1 (IRS-1) gene and demonstrated that they exhibited growth retardation and mild insulin resistance, suggesting the presence of IRS-1-independent pathway that partially substitutes for IRS-1 in IRS-1-deficient mice (Tamemoto, H., Kadowaki, T., Tobe, K., Yagi, T., Sakura, H., Hayakawa, T., Terauchi, Y., Ueki, K., Kaburagi, Y., Satoh, S., Sekihara, H., Yoshioka, S., Horikoshi, H., Furuta, Y., Ikawa, Y., Kasuga, M., Yazaki, Y., and Aizawa, S.(1994) Nature 372, 182-186). We have examined the [Medline] insulin-stimulated tyrosine-phosphorylated proteins in livers of wild type and IRS-1-deficient mice. Tyrosine phosphorylation of an 190-kDa protein (pp190) by insulin was significantly stimulated in livers of IRS-1-deficient mice, which was weakly observed in wild type mice in addition to IRS-1. We also demonstrated that pp190 was immunologically distinct from IRS-1 and was associated with both the 85-kDa subunit of phosphatidylinositol 3-kinase and the Grb2/Ash molecule as IRS-1. We identified pp190 as a novel substrate for insulin receptor kinase (IRS-2), which can bind both PI3-kinase and Ash/Grb2, and whose tyrosine phosphorylation is specifically induced in IRS-1-deficient mice. These data suggested that pp190 may play some physiological roles in insulin's signal transduction; furthermore, induction of tyrosine phosphorylation of pp190 may be one of the compensatory mechanisms that substitute for IRS-1 in IRS-1-deficient mice.


INTRODUCTION

Insulin induces a wide variety of growth and metabolic responses in many cell types. Insulin initiates its biological effects by activation of tyrosine kinase in the beta-subunit (Kasuga et al., 1983) and phosphorylates several proteins such as insulin receptor substrate-1 (IRS-1) (^1)(White et al., 1985; Kadowaki et al., 1987; Tobe et al., 1990), Shc (Pronk et al., 1993; Yonezawa et al., 1994), and pp60 (Momomura et al., 1988). These tyrosine-phosphorylated substrates bind several Src homology 2 proteins, thereby linking the tyrosine kinase to activation of cytoplasmic enzymes, which finally lead to the insulin's biological actions.

Among those substrates, IRS-1 has many tyrosine phosphorylation sites and is considered to be the platform of signaling complex. Tyrosine phosphorylation sites in IRS-1 provided binding sites for several distinct Src homology 2 proteins and may mediate multiple signaling pathways (Sun et al., 1991; White and Kahn, 1994). Indeed, IRS-1 binds the 85-kDa subunit of phosphatidylinositol 3-kinase (PI 3-kinase p85) through interaction with Tyr-X-X-Met motifs, thereby activating PI 3-kinase (Lavan et al., 1992; Backer et al., 1992; Myers et al., 1992; Folli et al., 1992). IRS-1 also binds Ash/Grb2 through interaction with Tyr-Val-Asn-IIe motif when tyrosine-phosphorylated, thereby activating p21leading to the activation of MAP kinase cascade (Tobe et al., 1993; Skolnik et al., 1993; Baltensperger et al., 1993; Matuoka et al., 1993). Moreover, IRS-1 has binding sites for Syp and Nck (Lee et al., 1993; Kuhne et al., 1993). With regard to other insulin receptor substrates, the oncoprotein Shc is tyrosine-phosphorylated on stimulation with insulin and also binds Ash/Grb2 (Pelicci et al., 1992; Pronk et al., 1993; Yonezawa et al., 1994). Therefore, IRS-1 and Shc may constitute two major distinct pathways in the signal transduction of insulin through Ash/Grb2. In addition, in adipocytes, a 60-kDa protein (pp60) is tyrosine-phosphorylated by insulin (Momomura et al., 1988). Recently, pp60 has been shown to bind PI 3-kinase (Lavan et al., 1993). Another substrate, IL-4-induced phosphotyrosine substrate (4PS), which is originally found in IL-3-dependent myeloid cell lines as a common substrate for IL-4 receptor and insulin receptor kinase and is similar in size to IRS-1, is reported to bind PI 3-kinase activity (Wang et al., 1993a, 1993b). The signal transduction of insulin through PI 3-kinase, therefore, may also have two pathways: IRS-1-dependent and IRS-1independent pathways.

To better understand the roles of IRS-1 in normal physiology, we and others generated mice with a targeted disruption of the IRS-1 gene locus (Tamemoto et al., 1994; Araki et al., 1994), and we demonstrated that IRS-1-deficient mice exhibited growth retardation and mild insulin resistance. These data suggested the existence of a pathway that can compensate for IRS-1 in IRS-1-deficient mice. In fact, we observed an insulin-induced increase in both PI 3-kinase activity and MAP kinase activity in livers of IRS-1-deficient mice (Tamemoto et al., 1994). These data urged us to examine the insulin-stimulated tyrosine-phosphorylated proteins in wild type and IRS-1deficient mice. Here, we report that tyrosine phosphorylation of a 190-kDa protein (pp190) induced by insulin was significantly increased in IRS-1-deficient mice. This pp190 was also observed, although less intensively, in wild type mice. Moreover, we demonstrated that pp190 binds both PI 3-kinase p85 and Ash/Grb2 molecule as IRS-1. These data suggested that pp190 may play some physiological roles in wild type mice and, furthermore, the pp190 pathway may serve as one of the compensatory mechanisms to substitute for IRS-1 in IRS-1-deficient mice.


EXPERIMENTAL PROCEDURES

Materials

PI (bovine liver) was purchased from Avanti Polar Lipids, Inc. Protein G-Sepharose was from Pharmacia Biotech Inc. [-P]ATP (6,000 Ci/mmol) was from DuPont NEN. Prestained molecular weight markers for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were from Bio-Rad. Porcine insulin was a gift from Eli Lilly Co. Actrapid human insulin was from Novo Nordisk A/S (Bagsvaerd, Denmark).

Antibodies

The anti-phosphotyrosine antibody (alphaPY:PY20) and alkaline-phosphatase-conjugated RC20A were purchased from Transduction Laboratories. The antibody against the 85-kDa subunit of PI 3-kinase, AB6, was from MBL (Nagoya, Japan). The antibody against Ash/Grb2 was from Santa Cruz Biotechnology, Inc. The anti-IRS-1 antibody (alphaIRS-1(1-6)) was a kind gift from Dr. Masaki Nishiyama (Jikei University School of Medicine, Tokyo) (Furusaka et al., 1994).

Mice

Male mice weighing about 20 g were used in the studies. Mice were fasted for at least 6 h before the experiment. The genotypes of the mice used in this experiments were determined as described (Tamemoto et al., 1994).

Immunoprecipitation Followed by Immunoblotting and PI 3-Kinase Assay

Mice were anesthetized by the administration of 2 mg of pentobarbital sodium intraperitoneally 10-15 min before the experiment. Portal veins were exposed, and 20 units of insulin was injected via portal veins. At 75 s, livers were removed, homogenized in ice-cold buffer A (25 mM Tris-HCl, pH 7.4, 10 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride) and centrifuged. The supernatants were incubated with PY20, alphaIRS-1(1-6), or alphaAsh/Grb2, followed by the addition of Protein G-Sepharose. The immunoprecipitates were washed with 1% Nonidet P-40-buffer A three times, were subjected to immunoblotting with RC20 followed by the detection by using an alkaline phosphatase conjugate system (Promega). The immunoprecipitates with alphaIRS-1(1-6) or alphaAsh/Grb2 were subjected to PI 3-kinase assay as described previously (Fukui et al., 1989).


RESULTS

Tyrosine Phosphorylation of pp190 Is Increased in Liver from Insulin-injected IRS-1-deficient Mice Compared with That in Wild Type Mice

Wild type or homozygous mice were injected with insulin via portal veins. 75 seconds after insulin injection, livers were removed, homogenized, and centrifuged. The supernatants were subjected to immunoprecipitation with alphaPY followed by immunoblotting with RC20A. We observed a 160-kDa tyrosine-phosphorylated protein in liver of insulin-injected wild type mice (Fig. 1, lane b). This 160-kDa tyrosine-phosphorylated protein was recognized by an anti-IRS-1 antibody (alphaIRS-1(1-6)) and was thus identified as IRS-1 (Fig. 1, lane c). In addition to IRS-1, we also observed another weakly tyrosine-phosphorylated protein of 190 kDa, which was not recognized by alphaIRS-1 (Fig. 1, lanec). In contrast, in homozygous IRS-1-deficient mice, we observed a 190-kDa tyrosine-phosphorylated protein (pp190) in insulin-injected mice (Fig. 1, lane e). The 190-kDa protein was not recognized by alphaIRS-1 (Fig. 1, lane f). We did not observe a 160-kDa tyrosine-phosphorylated protein in alphaIRS-1(1-6) immunoprecipitates (Fig. 1, lane f). Tyrosine phosphorylation of the 190-kDa protein was significantly increased in insulin-injected IRS-1-deficient mice compared with that in insulin-injected wild type mice (Fig. 1, lanes b and e). These data suggested that insulin-stimulated tyrosine phosphorylation of pp190, which was immunologically distinct from IRS-1, was significantly increased in IRS-1-deficient mice compared with that in wild type mice. We sometimes observed a 160-kDa protein, which was not recognized by alphaIRS-1 in insulin-injected IRS-1-deficient mice (Fig. 1, lane e), although the detection of this band is not reproducible.


Figure 1: Detection of pp190 in insulin-injected wild type or IRS-1-deficient mice. Wild type (lanes a-c) or IRS-1-deficient mice (lanes d-f) were injected without (lanesa and d) or with (lanes b, c, e, and f) insulin via portal veins. At 75 s, livers were removed, homogenized in buffer A, and centrifuged. The supernatants were subjected to immunoprecipitation with alphaPY (lanesa, b, d, and e) or alphaIRS-1(1-6) (lanes c and f), followed by Western blotting with RC20A.



pp190 Is Able to Bind Both PI 3-Kinase p85 and Ash/Grb2 as IRS-1

It has been shown that tyrosine-phosphorylated IRS-1 binds both p85 subunit of PI 3-kinase (p85) and Ash/Grb2 (Tobe et al., 1993; Skolnik et al., 1993). Therefore, we also examined whether pp190 binds p85 or Ash/Grb2 as IRS-1 does. The liver lysates from the wild type or homozygous mice without or with insulin injection were subjected to immunoprecipitation with alphap85 or alphaAsh/Grb2 followed by immunoblotting with RC20A.

In alphap85 immunoprecipitates in insulin-injected wild type mice livers, we observed a 160-kDa tyrosine-phosphorylated protein (Fig. 2, lane b), which is consistent with the previous observation that PI 3-kinase p85 binds tyrosine-phosphorylated IRS-1. We also observed a 190-kDa band, although the intensity of the band was weaker (Fig. 2, lane b). In contrast, in insulin-injected IRS-1-deficient mice, we observed a 190-kDa tyrosine-phosphorylated protein in alphap85 immunoprecipitates (Fig. 2, laned). We did not observe a 160-kDa tyrosine-phosphorylated protein (Fig. 2, laned). The amount of pp190 in alphap85 immunoprecipitates was increased in IRS-1-deficient mice compared with that in wild type mice (Fig. 2, lanes b and d).


Figure 2: PI 3-kinase 85 kDa subunit (p85alpha) binds tyrosine-phosphorylated pp190 in wild type or IRS-1-deficient mice. The immunoprecipitates by alphap85alpha (AB6) from the liver lysates of wild type (lanesa and b) or IRS-1-deficient mice (lanes c and d) without (lanes a and c) or with (lanes b and d) insulin injection were subjected to Western blotting with RC20A.



We also examined whether pp190 binds Ash/Grb2 molecule. In wild type mice we observed a 160-kDa tyrosine-phosphorylated protein and a 190-kDa weakly tyrosine-phosphorylated protein in alphaAsh/Grb2 immunoprecipitates (Fig. 3, lanesb and e). In contrast, in IRS-1-deficient mice, we observed a tyrosine-phosphorylated protein of 190 kDa in alphaAsh/Grb2 immunoprecipitates (Fig. 3, lanes d and f). We did not observe a 160-kDa tyrosine-phosphorylated protein (Fig. 3, lanes d and f). These data suggested that tyrosine-phosphorylated pp190 binds Ash/Grb2 molecule.


Figure 3: Ash/Grb2 binds tyrosine-phosphorylated pp190 in wild type or IRS-1-deficient mice. The immunoprecipitates by alphaAsh/Grb2 from the liver lysates of wild type (lanes a, b, and e) or IRS-1-deficient mice (lanesc, d, and f) without (lanes a and c) or with insulin injection (lanesb, d, e, and f) were subjected to Western blotting with RC20A.



pp190 Is Able to Bind Both PI 3-Kinase and Ash/Grb2 in Simultaneously as IRS-1

Next, to examine whether pp190 is able to bind p85 and Ash/Grb2 simultaneously, we measured an insulin-stimulated PI 3-kinase activity in alphaAsh/Grb2 immunoprecipitates in wild type or IRS-1-deficient mice (Fig. 4). In wild type mice, we observed a significant increase in PI 3kinase activity in alphaAsh/Grb2 immunoprecipitates (Fig. 4, laneb). We also observed a significant increase in PI 3-kinase activity in insulin-injected IRS-1-deficient mice (Fig. 4, laned), suggesting the presence of (a) protein(s) other than IRS-1 which binds PI 3-kinase activity and Ash/Grb2 simultaneously in an insulin-dependent manner like IRS-1. While we observed a significant increase in PI 3-kinase activity in alphaIRS-1(1-6) immunoprecipitates in wild type mice, we did not observe an insulin-induced increase in PI 3-kinase activity in alphaIRS-1(1-6) immunoprecipitates in IRS-1-deficient mice (data not shown).


Figure 4: Insulin stimulates PI 3-kinase activity in alphaAsh/Grb2 immunoprecipitates in wild type or IRS-1-deficient mice. The alphaAsh/Grb2 immunoprecipitates from the liver lysates of wild type (lanesa and b) or IRS-1-deficient mice (lanesc and d) without (lanesa and c) or with (lanesb and d) insulin injection were subjected to PI 3-kinase assay.




DISCUSSION

We and others generated mice with a targeted disruption of the IRS-1 gene locus and demonstrated that IRS-1-deficient mice exhibited growth retardation and mild insulin resistance (Tamemoto et al., 1994; Araki et al., 1994). We also demonstrated that insulin can significantly stimulate PI 3-kinase activity and MAP kinase activity, albeit to a lesser extent, in IRS-1-deficient mice. These data urged us to search for other substrates that might compensate for the function of IRS-1. In this study, we have compared the insulin-stimulated tyrosine-phosphorylated proteins in wild type and IRS-1-deficient mice. We have demonstrated that tyrosine phosphorylation of an M(r) 190-kDa protein was significantly increased in insulin-injected IRS-1-deficient mice. We also demonstrated that pp190 is able to bind PI 3-kinase p85 and Ash/Grb2 molecule as IRS-1, although they are immuologically distinct.

Our data have shown that tyrosine-phosphorylated pp190 induced by insulin was detectable not only in IRS-1-deficient mice liver but also in wild type mice liver in alphaPY, alphap85alpha, and alphaAsh/Grb2 immunoprecipitates. pp190 is not a differently processed form of IRS-1 gene, since mRNA of IRS-1 was totally absent in IRS-1-deficient mice. The presence of tyrosinephosphorylated pp190 in wild type mice suggested that pp190 may have some physiological roles mediating insulin or insulin-like growth factors' action. It is possible that the expression of pp190 is essential for the insulin or insulin-like growth factors' action in some tissues or in certain stages of development. It is also possible that the expression of IRS-1 and pp190 may be differentially regulated so that these two proteins have different biological roles. Further study such as the determination of primary structure of pp190 will be needed to clarify these points.

Despite the lack of IRS-1, insulin can significantly stimulate PI 3-kinase and MAP kinase activity in livers of IRS-1-deficient mice (Tamemoto et al., 1994). Since pp190 is the only tyrosine-phosphorylated protein detectable in IRS-1-deficient mice under our conditions, and pp190 seems to have the ability to couple insulin receptor kinase to a ras p21 pathway via Ash/Grb2-Sos complex and a PI 3-kinase pathway as IRS-1 does, induction of tyrosine phosphorylation of pp190 may explain the mechanisms, at least in part, how deficient IRS-1 was compensated in IRS-1-deficient mice. The importance of pp190 is further supported by the fact that tyrosine phosphorylation of pp190 is specifically induced in IRS-1-deficient mice. Nevertheless, we cannot exclude the possibility that Shc protein, pp60-like molecule, or 4PS may also contribute to the compensatory mechanisms, although a significant increase in the degree of insulin-stimulated tyrosine phosphorylation of Shc protein was not seen in IRS-1-deficient mice (Tamemoto et al., 1994), nor did we observe pp60 clearly under our conditions.

pp190 and IRS-1 have several characteristics in common as a substrate for insulin receptor kinase. First, tyrosine phosphorylation was induced within 30 s after insulin stimulation(data not shown). Second, both of them are able to bind PI 3-kinase and Ash/Grb2 simultaneously. Third, they are cytosolic proteins, which can be extracted without detergent. It may be possible that they may belong to the same gene family.

In this experiment, we detected pp190 both in wild type and homozygous mice after insulin stimulation more definitely than previously (Tamemoto et al., 1994). In our previous report, we homogenized the mice liver in a boiling homogenization buffer containing SDS. In contrast, we homogenized the mice liver in an ice-cold homogenization buffer in this report. It may be advantageous to use the ice-cold buffer without SDS to detect pp190, pp190bulletp85 complex, and pp190bulletGrb2/Ash complex.

It is of note that 4PS, which was originally found in IL-3-dependent FDC cell lines as a common substrate for IL-4 receptor and insulin receptor kinase with a molecular weight similar to that of IRS-1, is a 170-kDa protein that can bind PI 3-kinase activity (Wang et al., 1993a, 1993b). It is possible that pp190 may in fact be 4PS. However, pp190 is slightly higher in molecular weight than IRS-1 (Fig. 1), which has been reported to be similar in size to 4PS (Wang et al., 1993a, 1993b).


FOOTNOTES

*
This work was supported by Grant 190831 from the Juvenile Diabetes Foundation International (to T. K.) and by a grant for diabetes research from the Ohtsuka Pharmaceutical Co., Ltd. (to T. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence and reprint requests should be addressed: Third Dept. of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. Fax: 81-3-5689-7209 or 81-3-5684-3987.

(^1)
The abbreviations used are: IRS-1, insulin receptor substrate-1; PI, phosphatidylinositol; PI 3-kinase p85, phosphatidylinositol 3-kinase 85-kDa subunit; Ras, p21; MAP kinase, mitogen-activated protein kinase; Ash, abundant Src homology; Grb2, growth factor receptor-bound protein 2; IL, interleukin; 4PS, IL-4 phosphotyrosine substrate; IRS-2, insulin receptor substrate-2; alphaIRS-1, an anti-insulin receptor substrate-1 antibody; alphaPY, an anti-phosphotyrosine antibody; alphap85, an anti-p85 antibody; alphaAsh/Grb2, an anti-Ash/Grb2 antibody.


ACKNOWLEDGEMENTS

We thank Dr. Masato Kasuga for helpful discussions and critical reading of this manuscript. We thank Dr. Masaki Nishiyama (Jikei University School of Medicine) for the kind gift of an antibody against IRS-1(1-6). We thank Dr. Kinori Kosaka, Dr. Ryoko Hagura, Director Hajime Kawashima, and Dr. Osamu Koshio of the Institute for Diabetes Care and Research, Asahi Life Foundation. We also thank Drs. Yasushi Kaburagi, Kohjiro Ueki, Yoshihiko Takahashi, Shoji Asai, Tadashi Yamamoto, Noriko Takahashi, and Ritsuko Yamamoto-Honda for support.

Addendum-It seems likely that pp190, which we demonstrated in this report, is the same molecule as IRS-2, as reported by Araki et al.(1994).


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