(Received for publication, October 31, 1994; and in revised form, January 20, 1995)
From the
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.
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 -subunit (Kasuga et
al., 1983) and phosphorylates several proteins such as insulin
receptor substrate-1 (IRS-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.
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 PY (lanesa, b, d, and e) or
IRS-1(1-6) (lanes c and f), followed by Western
blotting with RC20A.
In p85
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
p85 immunoprecipitates (Fig. 2, laned). We did not observe a 160-kDa tyrosine-phosphorylated
protein (Fig. 2, laned). The amount of pp190
in
p85 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 (p85)
binds tyrosine-phosphorylated pp190 in wild type or IRS-1-deficient
mice. The immunoprecipitates by
p85
(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 Ash/Grb2 immunoprecipitates (Fig. 3, lanesb and e). In contrast,
in IRS-1-deficient mice, we observed a tyrosine-phosphorylated protein
of 190 kDa in
Ash/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
Ash/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.
Figure 4:
Insulin stimulates PI 3-kinase activity in
Ash/Grb2 immunoprecipitates in wild type or IRS-1-deficient mice.
The
Ash/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.
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 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 PY,
p85
, and
Ash/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, pp190p85 complex, and pp190
Grb2/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).
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).