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. 2004 Jul 19;166(2):213–223. doi: 10.1083/jcb.200403069

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Copyright © 2004, The Rockefeller University Press

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Figure 5. — IRS-1 is a novel S6K substrate. (A) Domain structure of IRS-1 indicating regions of GST fusions (aa numbering based on murine IRS-1) used for in vitro kinase assays and comparison of the S6 phosphorylation site with candidate S6-like phosphorylation sites present in IRS-1^108–516. Conserved arginines are shown in bold, serines phosphorylated by S6K in S6 and potentially phosphorylated in IRS-1^108–516 are shown in red, and serines mutated to alanines in mutants of sites 1–3 are underlined. Also shown is the homologous site to site 2 in murine IRS-2. (B) In vitro kinase assay of GST-IRS-1 fragments with purified S6K2. Full-length GST fusion proteins (1–4, as indicated in A) and 40S ribosomal proteins containing S6 (40S) in these preparations are indicated with asterisks in the Coomassie blue–stained gel (right), and the ³²P-labeled proteins after the kinase assay and autoradiography are shown on the left. Two phosphorylated bands are noted in kinase assay with IRS-1^108–516, the lower of which represents a poorly resolved doublet of cleaved forms of the protein. An arrow indicates ³²P-labeled S6 present in the 40S ribosome preparation. (C) In vitro kinase assay of IRS-1 site-directed mutants by S6K1. Top panels, autoradiographs; bottom panels, Coomassie blue–stained gels; left panels, phosphorylation of IRS-1^108–516 (WT) and site 1–3 compound mutants as indicated in Fig. 4 A; right panels, phosphorylation of IRS-1^108–516 and individual serine-to-alanine mutants of the serine residues within site 2. (D) Ser302 phosphorylation of IRS-1^108–516 wild-type (WT) or IRS-1^108–516 with Ser302 mutated to alanine (S302A) detected with a phosphospecific antibody to IRS-1 Ser302 after in vitro phosphorylation by S6K1. (E) IRS-1 Ser302 phosphorylation is elevated in vivo. Top, Ser302-phosphorylated IRS-1 detected by a phosphospecific Ser302 antibody in extracts from serum-starved isogenic TSC2 ^+/+ and TSC2 ^−/− MEFs, and TSC2 ^−/− MEFs lines in which wild-type (−/− (+WT)) or a pathogenic mutant TSC2 (−/− (+N1643K)) has been reintroduced; top, Ser302-phosphorylated IRS-1; bottom, total IRS-1. (F) IRS-1 Ser302 phosphorylation is inhibited by mTOR/S6K inhibition with rapamycin in serum-starved TSC2 ^−/− MEFs. Top, Ser302-phosphorylated IRS-1; bottom, total IRS-1. Where indicated, MEFs were also stimulated with insulin for 10 min. (G) IRS-1 Ser302 phosphorylation is diminished in TSC2 ^−/− MEFs after siRNA-mediated inhibition of S6K1. Top, Ser302-phosphorylated IRS-1; bottom, total IRS-1. Where indicated, MEFs were also stimulated with insulin for 10 min.

Figure 5. — IRS-1 is a novel S6K substrate. (A) Domain structure of IRS-1 indicating regions of GST fusions (aa numbering based on murine IRS-1) used for in vitro kinase assays and comparison of the S6 phosphorylation site with candidate S6-like phosphorylation sites present in IRS-1^108–516. Conserved arginines are shown in bold, serines phosphorylated by S6K in S6 and potentially phosphorylated in IRS-1^108–516 are shown in red, and serines mutated to alanines in mutants of sites 1–3 are underlined. Also shown is the homologous site to site 2 in murine IRS-2. (B) In vitro kinase assay of GST-IRS-1 fragments with purified S6K2. Full-length GST fusion proteins (1–4, as indicated in A) and 40S ribosomal proteins containing S6 (40S) in these preparations are indicated with asterisks in the Coomassie blue–stained gel (right), and the ³²P-labeled proteins after the kinase assay and autoradiography are shown on the left. Two phosphorylated bands are noted in kinase assay with IRS-1^108–516, the lower of which represents a poorly resolved doublet of cleaved forms of the protein. An arrow indicates ³²P-labeled S6 present in the 40S ribosome preparation. (C) In vitro kinase assay of IRS-1 site-directed mutants by S6K1. Top panels, autoradiographs; bottom panels, Coomassie blue–stained gels; left panels, phosphorylation of IRS-1^108–516 (WT) and site 1–3 compound mutants as indicated in Fig. 4 A; right panels, phosphorylation of IRS-1^108–516 and individual serine-to-alanine mutants of the serine residues within site 2. (D) Ser302 phosphorylation of IRS-1^108–516 wild-type (WT) or IRS-1^108–516 with Ser302 mutated to alanine (S302A) detected with a phosphospecific antibody to IRS-1 Ser302 after in vitro phosphorylation by S6K1. (E) IRS-1 Ser302 phosphorylation is elevated in vivo. Top, Ser302-phosphorylated IRS-1 detected by a phosphospecific Ser302 antibody in extracts from serum-starved isogenic TSC2 ^+/+ and TSC2 ^−/− MEFs, and TSC2 ^−/− MEFs lines in which wild-type (−/− (+WT)) or a pathogenic mutant TSC2 (−/− (+N1643K)) has been reintroduced; top, Ser302-phosphorylated IRS-1; bottom, total IRS-1. (F) IRS-1 Ser302 phosphorylation is inhibited by mTOR/S6K inhibition with rapamycin in serum-starved TSC2 ^−/− MEFs. Top, Ser302-phosphorylated IRS-1; bottom, total IRS-1. Where indicated, MEFs were also stimulated with insulin for 10 min. (G) IRS-1 Ser302 phosphorylation is diminished in TSC2 ^−/− MEFs after siRNA-mediated inhibition of S6K1. Top, Ser302-phosphorylated IRS-1; bottom, total IRS-1. Where indicated, MEFs were also stimulated with insulin for 10 min.