Project

Project 3

Elucidation of mechanisms of modulation of IGF/insulin signals through insulin receptor, IGF-I receptor, and IRS family proteins

   Our ongoing investigations to identify proteins that interact with IRSs and elucidate their function in signal transduction of IGF/insulin are driven by accumulated data showing that IRS-associated proteins modulate IRS tyrosine phosphorylation induced by IGF or insulin and IRS formed >700 kDa protein complex (we call it IRSome).

   Firstly, By yeast two-hybrid screening, we identifyied an array of proteins that associate with IRSs not through recognition of phosphotyrosine, because yeast has few tyrosine kinases. Our two-hybrid system utilized full length IRS-1, IRS-2 or IRS-3 as a bait and placenta, adipocytes, or FRTL-5 cDNA libraries as a prey. One of the interacting proteins is diacylglycerol kinase (DGK) which catalyzes the phosphorylation of diacylglycerol to phosphatidic acid. Our recent results show that DGK changes the tyrosine phosphorylation of IRSs induced by IGF-I. Besides DGK, we found that TSPYL (testis-specific protein, Y-encoded-like protein) and IRSAL (IRS-associated LIM protein) also bind to IRS and especially IRSAL modulates IRS-1 and IRS-2 tyrosine phosphorylation induced by IGF or insulin. TSPYL is expressed ubiquitously in various tissues; however its function is unclear to date. IRSAL is a novel protein possessing a LIM domain that is known to be important for protein-protein interaction. In addition, we demonstrated that IRSs also bound to the protein composing AP (adaptor protein)-1 that is reported to play important roles in the sorting of proteins from the trans-Golgi network to the early endosomes. Based on our data, we hypothesized that IRSs are associated with AP-1 after its protein synthesis, and concentrated on the surface of the early endosomes, suggesting that adequate localization of IRS-1 in a specific cell compartment through these novel mechanisms is essential for insulin/IGF actions. In contrast, IRS-3 was localized not only adjacent to the plasma membrane but also in the nucleus. We then studied subcellular localization of deletion mutants and fragments of IRS-3 fused with GFP. We found that the middle region of the phosphotyrosine binding domain played an important role in nuclear localization. This region includes sequences that are unique to IRS-3. We investigated intracellular localization of other IRSs fused with GFP. GFP-IRS-1, GFP-IRS-2, and GFP-IRS-4 were mainly localized in the cytosol or plasma membranes. Chimeric protein, Gal4 DNA binding domain fused with IRS-3 C-terminal region, increased transcription of the reporter gene containing Gal4 binding site in human embryonic kidney 293 cells. These results suggest that intracellular localization of IRS-3 is determined by a different mechanism from other IRS proteins, and that IRS-3 possesses a transcription-regulating activity.

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Project 3
Elucidation of mechanisms of modulation of IGF/insulin signals through insulin receptor, IGF-I receptor, and IRS family proteins

Our ongoing investigations to identify proteins that interact with IRSs and elucidate their function in signal transduction of IGF/insulin are driven by accumulated data showing that IRS-associated proteins modulate IRS tyrosine phosphorylation induced by IGF or insulin and IRS formed >700 kDa protein complex (we call it IRSome). Firstly, By yeast two-hybrid screening, we identifyied an array of proteins that associate with IRSs not through recognition of phosphotyrosine, because yeast has few tyrosine kinases. Our two-hybrid system utilized full length IRS-1, IRS-2 or IRS-3 as a bait and placenta, adipocytes, or FRTL-5 cDNA libraries as a prey. One of the interacting proteins is diacylglycerol kinase (DGK) which catalyzes the phosphorylation of diacylglycerol to phosphatidic acid. Our recent results show that DGK changes the tyrosine phosphorylation of IRSs induced by IGF-I. Besides DGK, we found that TSPYL (testis-specific protein, Y-encoded-like protein) and IRSAL (IRS-associated LIM protein) also bind to IRS and especially IRSAL modulates IRS-1 and IRS-2 tyrosine phosphorylation induced by IGF or insulin. TSPYL is expressed ubiquitously in various tissues; however its function is unclear to date. IRSAL is a novel protein possessing a LIM domain that is known to be important for protein-protein interaction. In addition, we demonstrated that IRSs also bound to the protein composing AP (adaptor protein)-1 that is reported to play important roles in the sorting of proteins from the trans-Golgi network to the early endosomes. Based on our data, we hypothesized that IRSs are associated with AP-1 after its protein synthesis, and concentrated on the surface of the early endosomes, suggesting that adequate localization of IRS-1 in a specific cell compartment through these novel mechanisms is essential for insulin/IGF actions. In contrast, IRS-3 was localized not only adjacent to the plasma membrane but also in the nucleus. We then studied subcellular localization of deletion mutants and fragments of IRS-3 fused with GFP. We found that the middle region of the phosphotyrosine binding domain played an important role in nuclear localization. This region includes sequences that are unique to IRS-3. We investigated intracellular localization of other IRSs fused with GFP. GFP-IRS-1, GFP-IRS-2, and GFP-IRS-4 were mainly localized in the cytosol or plasma membranes. Chimeric protein, Gal4 DNA binding domain fused with IRS-3 C-terminal region, increased transcription of the reporter gene containing Gal4 binding site in human embryonic kidney 293 cells. These results suggest that intracellular localization of IRS-3 is determined by a different mechanism from other IRS proteins, and that IRS-3 possesses a transcription-regulating activity.


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