Substrate specificity of TOR complex 2 is determined by a ubiquitin-fold domain of the Sin1 subunit.

Hisashi Tatebe,Chojiro Kojima,David Richter,Tomoyuki Hatano,Tomomi Furuya,Kazuhiro Shiozaki,Saori Kataoka,Toshiya Yonekura,Kyoko Furuita,Shinichi Murayama

doi:10.7554/elife.19594

Abstract

The target of rapamycin (TOR) protein kinase forms multi-subunit TOR complex 1 (TORC1) and TOR complex 2 (TORC2), which exhibit distinct substrate specificities. Sin1 is one of the TORC2-specific subunit essential for phosphorylation and activation of certain AGC-family kinases. Here, we show that Sin1 is dispensable for the catalytic activity of TORC2, but its conserved region in the middle (Sin1CRIM) forms a discrete domain that specifically binds the TORC2 substrate kinases. Sin1CRIM fused to a different TORC2 subunit can recruit the TORC2 substrate Gad8 for phosphorylation even in the sin1 null mutant of fission yeast. The solution structure of Sin1CRIM shows a ubiquitin-like fold with a characteristic acidic loop, which is essential for interaction with the TORC2 substrates. The specific substrate-recognition function is conserved in human Sin1CRIM, which may represent a potential target for novel anticancer drugs that prevent activation of the mTORC2 substrates such as AKT.

Highlights

Sin1 (SAPK-interacting protein 1) was identified in a yeast two-hybrid screen as a protein that interacts with the stress-activated Spc1/Sty1 MAP kinase (MAPK) in the fission yeast Schizosaccharomyces pombe (Wilkinson et al, 1999)
This observation might be due to the reduced level of RICTOR in the absence of SIN1 (Frias et al, 2006), we tested whether absence of Sin1 affects the TOR complex 2 (TORC2) integrity in the fission yeast S. pombe, in which TORC2 is not essential for cell viability
Immunoprecipitation of the FLAG epitope-tagged Tor1 detected its association with the RICTOR ortholog Ste20 in wild-type, Dsin1, Dwat1 and Dbit61 cell lysate, indicating that Tor1 interacts with Ste20 in the absence of Sin1 or other TORC2 subunits (Figure 1A)

Summary

Introduction

Sin (SAPK-interacting protein 1) was identified in a yeast two-hybrid screen as a protein that interacts with the stress-activated Spc1/Sty MAP kinase (MAPK) in the fission yeast Schizosaccharomyces pombe (Wilkinson et al, 1999). Sin orthologs have been identified in diverse eukaryotic species through various screens. A partial cDNA clone encoding a human ortholog was isolated in a genetic screen for suppressors of the RAS function in budding yeast (Colicelli et al, 1991). Biochemical isolation of proteins binding to mammalian MAPK kinase kinase, MEKK2, identified a human Sin ortholog (Cheng et al, 2005). A Dictyostelium homolog RIP3 was isolated in a yeast two-hybrid screen for proteins interacting with mammalian Ha-Ras (Lee et al, 1999)

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Conclusion