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. 2001 Jan;107(2):181-9.
doi: 10.1172/JCI10934.

Insulin/IGF-1 and TNF-alpha stimulate phosphorylation of IRS-1 at inhibitory Ser307 via distinct pathways

L Rui  1 , V Aguirre, J K Kim, G I Shulman, A Lee, A Corbould, A Dunaif, M F White
Affiliations

Insulin/IGF-1 and TNF-alpha stimulate phosphorylation of IRS-1 at inhibitory Ser307 via distinct pathways

L Rui et al. J Clin Invest. 2001 Jan.

Abstract

Serine/threonine phosphorylation of IRS-1 might inhibit insulin signaling, but the relevant phosphorylation sites are difficult to identify in cultured cells and to validate in isolated tissues. Recently, we discovered that recombinant NH2-terminal Jun kinase phosphorylates IRS-1 at Ser307, which inhibits insulin-stimulated tyrosine phosphorylation of IRS-1. To monitor phosphorylation of Ser307 in various cell and tissue backgrounds, we prepared a phosphospecific polyclonal antibody designated alphapSer307. This antibody revealed that TNF-alpha, IGF-1, or insulin stimulated phosphorylation of IRS-1 at Ser307 in 3T3-L1 preadipocytes and adipocytes. Insulin injected into mice or rats also stimulated phosphorylation of Ser307 on IRS-1 immunoprecipitated from muscle; moreover, Ser307 was phosphorylated in human muscle during the hyperinsulinemic euglycemic clamp. Experiments in 3T3-L1 preadipocytes and adipocytes revealed that insulin-stimulated phosphorylation of Ser307 was inhibited by LY294002 or wortmannin, whereas TNF-alpha-stimulated phosphorylation was inhibited by PD98059. Thus, distinct kinase pathways might converge at Ser307 to mediate feedback or heterologous inhibition of IRS-1 signaling to counterregulate the insulin response.

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Figures

Figure 1
Figure 1
Ser307 in IRS-1 mediates the inhibitory effect of anisomycin on insulin-induced tyrosyl phosphorylation of IRS-1. CHO cells were transiently transfected with plasmids (10 μg) encoding IRS-1 or IRS-1S307A. Twenty-four hours later, cells were deprived of serum overnight and preincubated for 30 minutes with 5 μg/ml anisomycin before 50 nM insulin stimulation for an additional 5 minutes. (a) Proteins (30 μg) in cell lysates were resolved by SDS-PAGE and immunoblotted with anti-phosphotyrosine. (b) The same blot was reprobed with αIRS-1. The migration of molecular weight standards, IRS-1, and IR is indicated.
Figure 2
Figure 2
Anisomycin stimulates phosphorylation of IRS-1 on Ser307. (a and b) CHO (a) or 293 (b) cells were treated with 5 μg/ml anisomycin (aniso) for 30 minutes. IRS-1 was immunoprecipitated (IP) with αIRS-1 and immunoblotted with αpS307. The same blot was stripped and reprobed with αIRS-1. (c) αpS307 was preincubated on ice for 15 minutes with 5-μg phospho-Ser307 peptide (pS307) or non-phospho-Ser307 (S307) peptide. αIRS-1 immunoprecipitates from control or anisomycin-stimulated CHO cells were immunoblotted with αpS307 in the presence of pS307 or S307, respectively. (d) CHO cells were transfected transiently with 5-μg plasmids encoding HA-tagged IRS-1 or IRS-1S307A. Cells were treated with 5 μg/ml anisomycin for 30 minutes. HA-tagged IRS-1 and IRS-1S307A in cell lysates were immunoprecipitated with αHA. The αHA immunoprecipitates were divided evenly into two and immunoblotted with αpS307 and αIRS-1, respectively.
Figure 3
Figure 3
TNF-α stimulates phosphorylation of IRS-1 on Ser307 and inhibits insulin-promoted tyrosyl phosphorylation of IRS-1 and activation of ERK1/2. (a) 3T3-L1 adipocytes were pretreated with 20 ng/ml TNF-α for 5 hours before 50 nM insulin stimulation for 10 minutes. Proteins (40 μg) in cell lysates were immunoblotted with anti-phosphotyrosine and anti–phospho-MAPK, respectively. (b and c) 3T3-L1 preadipocytes (b) and adipocytes (c) were stimulated with 20 ng/ml TNF-α for 30 minutes. IRS-1 in cell lysates was immunoprecipitated with αIRS-1 and immunoblotted with αpS307. The same blots were stripped and reprobed with αIRS-1. (d) The 3T3-L1 adipocytes were stimulated with 20 ng/ml TNF-α for the indicated time. IRS-1 immunoprecipitates were immunoblotted with αpS307.
Figure 4
Figure 4
Insulin and IGF-1 stimulate phosphorylation of IRS-1 on Ser307 in both 3T3-L1 preadipocytes and adipocytes. (a) 3T3-L1 preadipocytes were stimulated with 100 nM insulin for the indicated time. IRS-1 in cell lysates was immunoprecipitated with αIRS-1 and immunoblotted with αpS307. The same blot was stripped and reprobed with αIRS-1. (b) 3T3-L1 preadipocytes and adipocytes were stimulated for 30 minutes with 100 nM insulin (Ins) and 100 ng/ml IGF-1 (IGF), respectively. IRS-1 immunoprecipitates were immunoblotted with αpS307. The same blot was stripped and reprobed with αIRS-1.
Figure 5
Figure 5
Insulin promotes phosphorylation of IRS-1 on Ser307in mouse, rat, and human skeletal muscle. (a) Saline or 5 U human insulin was injected as a bolus into the inferior vena cava of anesthetized mice. Soleus muscle was isolated 5 minutes later and homogenized. IRS-1 was immunoprecipitated with αIRS-1 and immunoblotted with αpS307. The same blots were stripped and reprobed with αIRS-1. (b) Insulin (5 U) was injected intraperitoneally into anesthetized rat. Saline was used as control. Soleus muscle was isolated 5 minutes later and homogenized. IRS-1 immunoprecipitates were immunoblotted with αpS307 or αIRS-1. Phospho-Ser307 was quantitated by phosphoimaging and normalized to IRS-1 level. The data were presented as mean ± SEM (three rats for each group; AP < 0.02). (c) Normal volunteer subjects were subjected to hyperinsulinemic euglycemic clamp. Muscle biopsies were obtained after insulin-clamp for 0 or 15 minutes and homogenized. IRS-1 immunoprecipitates were immunoblotted with αpS307. The data represent one of three independent experiments.
Figure 6
Figure 6
Two distinct pathways mediate Ser307 phosphorylation on IRS-1 induced by insulin/IGF-1 and TNF-α. (a) The 3T3-L1 preadipocytes were preincubated for 30 minutes with 20 μM LY294002 or 100 nM wortmannin (Wort) before stimulation for an additional 30 minutes with 100 nM insulin, 100 ng/ml IGF-1, or 20 ng/ml TNF-α. Proteins (50 μg) in the cell lysates were immunoblotted directly with antibodies against phospho-Akt or phospho-MAPK; or IRS-1 was immunoprecipitated from the lysates with αIRS-1 and immunoblotted with αpS307 or with αIRS-1. (b) The 3T3-L1 preadipocytes were preincubated for 30 minutes with 100 μM PD98059 before stimulation for an additional 30 minutes with 100 nM insulin, 100 ng/ml IGF-1, or 20 ng/ml TNF-α. IRS-1 in cell lysates was immunoprecipitated with αIRS-1. Half of the αIRS-1 immunoprecipitates were immunoblotted with αpS307, and half were immunoblotted with αIRS-1. (c) The 3T3-L1 preadipocytes were preincubated for 30 minutes with the indicated concentration of PD98059 before stimulation for an additional 30 minutes with 20 ng/ml TNF-α. Immunopurified IRS-1 was immunoblotted with αpS307. Cell extracts were immunoblotted with anti–phospho-MAPK.
Figure 7
Figure 7
TNF-α and insulin act synergistically to promote phosphorylation of Ser307 in IRS-1. The 3T3-L1 preadipocytes were treated for 30 minutes with 20 ng/ml TNF-α and 100 nM insulin either separately or simultaneously. IRS-1 in cell lysates was immunoprecipitated with αIRS-1 and immunoblotted with αpS307.
Figure 8
Figure 8
Kinase(s) other than JNK phosphorylate Ser307 in IRS-1. (a) The 3T3-L1 preadipocytes were preincubated for 30 minutes with or without 100 μM PD98059 before stimulation for an additional 15 minutes with 20 ng/ml TNF-α. JNK-1 was immunoprecipitated with αJNK-1, and subjected to an in vitro kinase assay using GST-cJun as a substrate (top). Cell lysates were immunoblotted with anti–phospho-MAPK (bottom). (b) The 3T3-L1 preadipocytes were preincubated for 30 minutes with or without 20 μM LY294002 before stimulation for an additional 10 minutes with 100 nM insulin. JNK-1 was immunoprecipitated with αJNK1, and subjected to an in vitro kinase assay using GST-cJun as a substrate.
Figure 9
Figure 9
Models for the function of Ser307 phosphorylation. Upon insulin stimulation, IRS-1 is tyrosyl-phosphorylated by the IR, resulting in the activation of PI 3-kinase that mediates Ser307 phosphorylation. Ser307 phosphorylation subsequently inhibits the ability of IRS-1 to be further tyrosyl phosphorylated by the IR and to propagate insulin signaling. An increase in Ser307 phosphorylation, such as that possibly trigged by TNF-α in chronic obesity, also induces insulin resistance. As β cells compensate for insulin insensitivity with compensatory hyperinsulinemia, chronic insulin stimulation induces more Ser307 phosphorylation through the PI 3-kinase pathway, thus further increasing the pool of inactive, Ser307 phosphorylated IRS-1. This vicious cycle might continue until hyperinsulinemia fails to compensate for insulin resistance.
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