Stress activated protein kinase (SAPK) interacting protein 1 (Sin1) is a member of a recently characterized gene family, conserved from yeast to humans. The gene copy number is strictly conserved (one Sin1 gene per genome), and the protein may be expressed ubiquitously in mammalian tissues. The Sin1 family has been implicated in several different signal transduction pathways. Originally identified as a partial cDNA and candidate Ras inhibitor, recent functional studies have revealed interactions with an interferon (IFN) receptor subunit (IFNAR2), and the SAPK JNK. Interactions have also been described between the yeast orthologues and the phosphatidylinositol kinase TOR2. Collectively, these data suggest that Sin1 has an important cellular role, and this study has investigated possible functions for this protein. As human Sin1 proteins have no paralogues within the genome, secondary structure homology was used to identify major domains within the protein. Four major domains within human Sin1 were deduced: an N-terminal domain containing a functional nuclear localization signal, a functional nuclear export signal, and a coiledcoil region; the conserved region in the middle that is likely to be a ubiquitin-like β-grasp protein binding domain; a Ras binding domain; and a pleckstrin homology-like domain that targets Sin1 to the plasma membrane and lipid rafts in vivo. Full and partial length EGFP constructs were used to examine the localization of human Sin1, and several isoforms derived from alternative splicing. All isoforms localized to the nucleus and nucleolus. Beyond this, Sin1α and Sin1ϒ had cytoplasmic staining, while Sin1 and Sin1β were also found at the plasma membrane and lipid rafts. Both the N-terminal domain and the conserved region in the middle were found to contribute to nuclear localization. Comparative genomic analysis between human, mouse, rat, dog, and chicken Sin1 genes revealed a number of conserved intronic regions, and the putative functions of these were predicted. Additionally, a putative promoter module within a CpG island and encompassing the transcription start site was predicted in all species. The human CpG island was found to have promoter activity in HEK293 cells. Using bioinformatics, genes that may be co-regulated with Sin1 were identified. These genes contained the Sin1 promoter module, and were found to co-express in large scale gene expression studies. Most of these genes were directly involved in the cellular response to pathogen infection, suggesting a conserved role for Sin1 in this pathway. Key biochemical functions of the Sin1 proteins were also identified, including the ability of Sin1 proteins to form dimers, and the ability of over-expressed Sin1 to induce apoptosis (mediated through the conserved region in the middle). Additionally, endogenous Sin1 protein levels were found to change following serum deprivation and hypoosmotic stress. Together, these studies have provided significant insight into the cellular role of Sin1, suggesting a role in inducing apoptosis as part of the IFN response to viral infection. The biological significance of the Sin1 proteins is discussed in the context of their predicted functions and the evolution of the protein family.

TRIM7 is a member of the TRIM (tripartite motif-containing) protein superfamily. This family has been implicated in many disorders such as genetic diseases, neurological diseases, and cancers. Little is known about the function of TRIM7 except that it interacts with glycogenin and may regulate glycogen biosynthesis. Recently, a yeast two-hybrid protein-protein interaction screen revealed the binding of TRIM7 to Sin1, a protein found in a complex with the mammalian target of rapamycin (mTOR) protein kinase. mTOR can form two complexes, mTORC1 and mTORC2, which are important for cell growth, differentiation, and survival. Sin1 is a core component of mTORC2 and is critical for mTORC2 stability and activity. It was confirmed by co-immunoprecipitation that TRIM7 associates with Sin1 and mTOR in cultured mammalian cells. Furthermore, it was demonstrated that TRIM7 is a phosphoprotein, although it was not directly targeted by mTOR in vitro. Similar to some other TRIM family proteins, it was demonstrated that TRIM7 has a ubiquitin E3 ligase function allowing it to autoubiquitinate both in vitro and in cells. The autoubiquitination of TRIM7 was dependent on its RING domain. Further characterization of TRIM7 indicated that it can both homo-oligomerise as well as hetero-oligomerise with other members of its sub-class of TRIM proteins and that it co-localises with them into discrete cytoplasmic loci. To determine the cellular function of TRIM7, a stable cell line expressing an shRNA directed against TRIM7 was generated. Successful knock down of TRIM7 was achieved and this led to an increase in the protein levels of components of the mTORC2 complex, including Sin1. This coincided with an increase in cell proliferation. In conclusion, this research identifies a novel role for TRIM7 as a ubiquitin ligase involved in regulating cell proliferation and provides a potential link between TRIM7 and the mTOR pathway, a major transducer of proliferative and cell survival signals.