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Structural and biochemical characterization of the KRLB region in insulin receptor substrate-2

Nature Structural & Molecular Biology volume 15, pages 251–258 (2008)Cite this article

Abstract

Insulin receptor substrates 1 and 2 (IRS1 and -2) are crucial adaptor proteins in mediating the metabolic and mitogenic effects of insulin and insulin-like growth factor 1. These proteins consist of a pleckstrin homology domain, a phosphotyrosine binding domain and a C-terminal region containing numerous sites of tyrosine, serine and threonine phosphorylation. Previous yeast two-hybrid studies identified a region unique to IRS2, termed the kinase regulatory-loop binding (KRLB) region, which interacts with the tyrosine kinase domain of the insulin receptor. Here we present the crystal structure of the insulin receptor kinase in complex with a 15-residue peptide from the KRLB region. In the structure, this segment of IRS2 is bound in the kinase active site with Tyr628 positioned for phosphorylation. Although Tyr628 was phosphorylated by the insulin receptor, its catalytic turnover was poor, resulting in kinase inhibition. Our studies indicate that the KRLB region functions to limit tyrosine phosphorylation of IRS2.

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Figure 1: Schematic diagram of IRS2.
Figure 2: Crystal structure of the KRLBY628–IRK complex.
Figure 3: In vitro binding of the KRLB region to IRK and IGF1RK.
Figure 4: Role of Tyr621 in the high Km(ATP) for Tyr628 phosphorylation.
Figure 5: Inhibition of IRK substrate phosphorylation by KRLBY628 and KRLBpY628.
Figure 6: Tyrosine phosphorylation of IRS2 in CHO-IR cells.

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Acknowledgements

This work was supported by the US National Institutes of Health (NIH) grant DK052916 (to S.R.H.) and a Howard Hughes Medical Institute (HHMI) Research Training Fellowship for Medical Students (to Y.D.T.). M.F.W. is an investigator at the HHMI. We thank W. Li and W.T. Miller (Stony Brook University, New York, USA) for purified IGF1RK, and W.T. Miller for manuscript comments. Beamline X4A at the National Synchrotron Light Source, Brookhaven National Laboratory, a US Department of Energy facility, is supported by the New York Structural Biology Consortium. The New York University Protein Analysis Facility is supported by NIH Shared Instrumentation Grant S10 RR017990, National Institute of Neurological Disorders and Stroke grant P30 NS050276 and US National Cancer Institute core grant P30 CA016087 (to T.A.N.).

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Authors and Affiliations

  1. and Department of Pharmacology, Structural Biology Program, Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, 540 First Avenue, New York, 10016, New York, USA

    Jinhua Wu, Chong-Feng Xu, Thomas A Neubert & Stevan R Hubbard

  2. Division of Endocrinology, Howard Hughes Medical Institute, Children's Hospital Boston, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Yolanda D Tseng & Morris F White

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  1. Jinhua Wu
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  2. Yolanda D Tseng
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  3. Chong-Feng Xu
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  4. Thomas A Neubert
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  5. Morris F White
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  6. Stevan R Hubbard
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Contributions

J.W. designed and performed the crystallographic studies, the in vitro biochemical experiments and a portion of the in-cell phosphorylation experiments, and contributed to manuscript preparation. Y.D.T. designed and performed a portion of the in-cell phosphorylation experiments and contributed to manuscript preparation. C.-F.X. acquired the MS data and contributed to manuscript preparation. T.A.N. supervised the MS experiments and contributed to manuscript preparation. M.F.W. designed and supervised the in-cell biochemical experiments and contributed to manuscript preparation. S.R.H. supervised the project and was the principal manuscript author.

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Correspondence to Stevan R Hubbard.

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Wu, J., Tseng, Y., Xu, CF. et al. Structural and biochemical characterization of the KRLB region in insulin receptor substrate-2. Nat Struct Mol Biol 15, 251–258 (2008). https://doi.org/10.1038/nsmb.1388

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