(Received for publication, July 25, 1995; and in revised form, August 21, 1995)
From the
Insulin receptor substrate-1 (IRS-1) is the major cytoplasmic substrate of the insulin and insulin-like growth factor (IGF)-1 receptors. Transgenic mice lacking IRS-1 are resistant to insulin and IGF-1, but exhibit significant residual insulin action which corresponds to the presence of an alternative high molecular weight substrate in liver and muscle. Recently, Sun et al. (Sun, X.-J, Wang, L.-M., Zhang, Y., Yenush, L. P., Myers, M. G., Jr., Glasheen, E., Lane, W. S., Pierce, J. H., and White, M. F. (1995) Nature 377, 173-177) purified and cloned 4PS, the major substrate of the IL-4 receptor-associated tyrosine kinase in myeloid cells, which has significant structural similarity to IRS-1. To determine if 4PS is the alternative substrate of the insulin receptor in IRS-1-deficient mice, we performed immunoprecipitation, immunoblotting, and phosphatidylinositol (PI) 3-kinase assays using specific antibodies to 4PS. Following insulin stimulation, 4PS is rapidly phosphorylated in liver and muscle, binds to the p85 subunit of PI 3-kinase, and activates the enzyme. Insulin stimulation also results in the association of 4PS with Grb 2 in both liver and muscle. In IRS-1-deficient mice, both the phosphorylation of 4PS and associated PI 3-kinase activity are enhanced, without an increase in protein expression. Immunodepletion of 4PS from liver and muscle homogenates removes most of the phosphotyrosine-associated PI 3-kinase activity in IRS-1-deficient mice. Thus, 4PS is the primary alternative substrate, i.e. IRS-2, which plays a major role in physiologic insulin signal transduction via both PI 3-kinase activation and Grb 2/Sos association. In IRS-1-deficient mice, 4PS/IRS-2 provides signal transduction to these two major pathways of insulin signaling.
Stimulation of the insulin and IGF-1 ()receptor
tyrosine kinases results in rapid autophosphorylation and subsequent
phosphorylation of cytoplasmic substrates. A major substrate of the
insulin receptor is IRS-1, a cytoplasmic protein of 160-185 kDa
on SDS-PAGE(1, 2, 3) . Following
insulin/IGF-1 stimulation, IRS-1 is rapidly phosphorylated on multiple
tyrosines(4) . This results in docking of several SH2 domain
proteins, including: the p85 subunit of PI
3-kinase(5, 6, 7, 8) , an upstream
element in insulin-stimulated glucose transport and activation of p70
S6 kinase(9, 10) ; Grb 2, an adapter molecule linking
IRS-1 to activation of Ras and mitogen-activated protein
kinase(11, 12, 13) ; and the tyrosine
phosphatase SHPTP2(14, 15) . Insulin and IGF-1
receptors can also phosphorylate other cytoplasmic proteins. These
include Shc, a cytoplasmic protein which binds to Grb 2(16) , a
p62 protein which associates with Ras-GAP(17) , and a
55-60-kDa protein which associates with PI
3-kinase(18, 19) .
Abundant evidence from Xenopus oocytes(20) , cell culture
systems(21, 22, 23) , and animal models (24, 25) has demonstrated the central role of IRS-1 in
mediating downstream effects of insulin and IGF-1. Recently, we (26) and others (27) have shown that mice made
IRS-1-deficient by targeted gene knockout exhibit hyperinsulinemia,
glucose intolerance, and marked growth retardation. However,
IRS-1-deficient mice continue to exhibit some insulin-stimulated
glucose disposal and phosphotyrosine-associated PI 3-kinase activation,
suggesting the presence of an IRS-1-independent pathway of signaling.
Immunoblots from both liver and muscle tissue of IRS-1
(-/-) animals reveal a 180-kDa protein (tentatively
designated IRS-2) which is tyrosine-phosphorylated within 1 min of
insulin stimulation and binds to PI 3-kinase, but is not immunoreactive
with anti-IRS-1 antibodies (26) .
A candidate protein for
IRS-1-independent signal transduction is 4PS, a protein of 180 kDa
initially observed as the primary substrate of the interleukin 4
receptor-associated tyrosine kinase(28) . In myeloid progenitor
cells, 4PS is rapidly phosphorylated in response to IL-4 or insulin,
binds p85, and activates PI 3-kinase. In myeloid cells which lack 4PS,
overexpression of IRS-1 can restore sensitivity to IL-4 and
insulin(29) . This functional similarity between 4PS and IRS-1
has been confirmed by the recent cloning of 4PS, which reveals an
IRS-1-like molecule with multiple conserved tyrosine phosphorylation
sites, as well as several homologous domains near the NH
terminus(30) . The similarity between these properties of 4PS
and the alternative substrate in IRS-1-deficient mice, as well as the
fact that IL-4 action is normal in IRS-1 (-/-) animals, (
)suggested to us that 4PS might be the alternative
substrate for insulin action in the IRS-1-deficient mouse.
Figure 1: Insulin-stimulated phosphorylation of 4PS/IRS-2 in the wild type (IRS-1 +/+) and knockout (IRS-1 -/-) mouse. A, phosphotyrosine immunoblots of IRS-1 and 4PS/IRS-2 immunoprecipitates. Extracts of liver or muscle from mice treated with diluent or insulin in vivo or myeloid precursor FDC-P2 cells treated with insulin in vitro were immunoprecipitated with anti-IRS-1 COOH-terminal (upper panel) or anti-4PS/IRS-2 (lower panel) antibody, separated by SDS-PAGE, and immunoblotted with anti-phosphotyrosine antibodies. The positions of IRS-1 and 4PS/IRS-2 are indicated by the arrows. The migration of phosphorylated 4PS from liver homogenates was similar to that of 4PS from insulin-treated FDC-P2 cells; the migration of phosphorylated 4PS in muscle was slightly more retarded, suggesting differences in phosphorylation state. For liver and muscle, each lane represents tissue from one animal; each blot is representative of at least three independent experiments. B, quantitation of 4PS/IRS-2 phosphorylation from phosphotyrosine immunoblots of anti-4PS/IRS-2 precipitates. Insulin-stimulated phosphorylation in wild type IRS-1 (+/+) animals was assigned a relative value of 100%. Data are mean ± S.E. for four independent experiments. C, anti-4PS/IRS-2 immunoblot of 4PS/IRS-2 immunoprecipitates. Supernatants of liver homogenates from mice treated with diluent or insulin in vivo were immunoprecipitated with anti-4PS/IRS-2, separated by SDS-PAGE, and immunoblotted with anti-4PS/IRS-2 antibodies.
Figure 2:
Insulin-stimulated association of
4PS/IRS-2 with the p85 subunit of PI 3-kinase and activation of PI
3-kinase activity. A, phosphotyrosine Western blot of anti-p85
immunoprecipitates from liver of wild type (IRS-1 +/+) and
knockout (IRS-1 -/-) mice. The migration of IRS-1 and IRS-2
is indicated by the arrows. B, p85 Western blot of
anti-4PS/IRS-2 immunoprecipitates from liver and muscle of wild type
and knockout mice. C, IRS-1, phosphotyrosine, and
4PS/IRS-2-associated PI 3-kinase activity in liver (upper
panel) and muscle (lower panel) of wild type and knockout
mice. Tissue homogenates were immunoprecipitated with anti-IRS-1
COOH-terminal, anti-4PS/IRS-2, or anti-phosphotyrosine 4G10, followed
by PI 3-kinase assays. P incorporation into PI 3-phosphate
was quantified using a PhosphorImager. Data are the mean ± S.E.
of three independent experiments and are expressed as activity relative
to the insulin-stimulated wild type animals (assigned a value of 100%).
*, p < 0.05;**, p <
0.001.
Direct immunoblots of anti-4PS precipitates revealed that p85 also associated with 4PS in an insulin-stimulated fashion in both liver and muscle (Fig. 2B). Again, p85 association with 4PS was enhanced in the IRS-1-deficient mice despite equivalent total p85 protein expression (data not shown). Precipitation of the same extracts with anti-4PS antibodies showed that insulin also stimulated PI 3-kinase activity in anti-4PS immunocomplexes in both wild type and knockout animals (Fig. 2C, right). The magnitude of the 4PS-associated PI 3-kinase activation was 55 and 141% greater in liver and muscle, respectively, of knockout than in control animals, consistent with the enhanced phosphorylation of 4PS in the absence of IRS-1.
Figure 3: Sequential immunodepletion of 4PS/IRS-2 from tissue homogenates: A and B, anti-phosphotyrosine 4G10 immunoblot of liver (A) or muscle (B) homogenates from insulin-stimulated mice subjected to four rounds of sequential anti-4PS/IRS-2 immunoprecipitation followed by anti-p85 immunoprecipitation as described under ``Experimental Procedures.'' The migration of residual IRS-2 (in both wild type and IRS-1 knockout animals) and of residual IRS-1 (in wild type animals only) is indicated by the arrows. C, PI 3-kinase activity with 4PS/IRS-2 immunodepletion in muscle of insulin-stimulated wild type and knockout mice. PI 3-kinase activity was measured in both anti-IRS-2 (left panels) and anti-phosphotyrosine (right panels) immune complexes following each of four successive immunoprecipitations with anti-4PS/IRS-2 antibody. Data are the means of duplicate assays performed after each immunodepletion step and are expressed in arbitrary PhosphorImager units.
Figure 4: Insulin-stimulated association of Grb 2 with IRS-1, IRS-2, and Shc in vivo. A, phosphotyrosine immunoblots of anti-Grb 2 immunoprecipitates from liver of wild type (IRS-1 +/+) and knockout (IRS-1 -/-) mice stimulated in vivo with insulin for 1.5 min. The migration of IRS-1 and IRS-2 is indicated by the arrows. B, anti-Shc immunoblot of anti-Grb 2 immunoprecipitates from liver of wild type and knockout mice stimulated in vivo with insulin for 1.5 min. The migration of the 52-kDa Shc isoform is indicated by the arrow.
The present study also shows that 4PS/IRS-2 may play a major role in early steps of insulin signal transduction in normal target tissues of intact animals. The exact contribution of 4PS/IRS-2 in comparison with IRS-1 in propagating the downstream insulin signal is difficult to determine due to the differing efficiency of immunoprecipitation with antibodies to IRS-1 and 4PS/IRS-2. Given the 50% reduction in insulin-stimulated glucose disposal in the IRS-1-deficient mice in vivo and decreased phosphotyrosine-associated PI 3-kinase activity in muscle(26) , we would predict that 4PS/IRS-2 may contribute up to 50% of total insulin-stimulated activation of PI 3-kinase and downstream glucose transport in skeletal muscle. The enhanced phosphotyrosine-associated PI 3-kinase activity observed in liver relative to muscle, despite similar magnitude of insulin stimulation of 4PS/IRS-2-associated PI 3-kinase, may reflect a greater abundance of 4PS/IRS-2 protein in liver as compared with muscle (data not shown) or, alternatively, the presence of additional phosphoproteins which may play a role in insulin signaling in liver. Although we have been unable to detect additional phosphoproteins in either liver or muscle thus far, we cannot entirely exclude their presence given limitations in sensitivity at the protein level. However, IRS-1-deficient mice show no difference in insulin-stimulated Shc phosphorylation and exhibit no other phosphoproteins, such as the p55-p60 proteins described by others(18, 19) , which bind to or activate PI 3-kinase (data not shown).
In summary, 4PS, initially identified as the IL-4 receptor-associated substrate, plays a role in normal insulin signaling in physiologically relevant tissues and helps to rescue the IRS-1-deficient mouse. Thus, 4PS/IRS-2 is a common substrate of two physiologically diverse growth factor receptors. Further investigation will attempt to clarify how specificity for metabolic versus cytokine downstream signaling is achieved.