Mutations in TSC1 and TSC2 are involved in the development of tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM). These two proteins are thought to function as a heterodimer, where TSC1 stabilises TSC2 and enhances its GTPase-activating protein (GAP) activity. It is this activity which stimulates the small GTPase Rheb, switching it from its mTORC1-activating (GTP-bound) state to its inactive (GDP-bound) state. For more information, do look at our signalling diagram for a simplified illustration of these interactions. A recent study by Dibble et al. (2012) has now identified TBC1 domain family member 7 (TBC1D7) as a component of the TSC1-TSC2 complex, and has implicated it in the regulation of mTORC1 signalling.
Mass spectroscopy was used to identify proteins which immunoprecipitated with FLAG-tagged TSC1 and TSC2 from HEK293 cell lysates. Using this method, TBC1D7 was seen to be the most abundant interacting protein detected from two independent runs. Co-immunoprecipitation experiments confirmed that endogenous TBC1D7 associated with the TSC1-TSC2 complex in lysates from HeLa cells and many different mouse tissues. TSC1 immunoprecipitation followed by a number of high salt washes also demonstrated that TBC1D7 is tightly bound to the TSC1-TSC2 complex in HeLa cell lysates.
Further co-immunoprecipitation studies showed that siRNA-mediated knockdown of TBC1D7 led to a reduction in the association between TSC1 and TSC2 in HeLa cells. However, siRNA knockdown of TBC1D7 and TSC1 did not affect the late endosomal/lysosomal localisation of TSC2 in serum-starved HeLa cells. The authors did note that stable shRNA knockdown of TBC1D7 in HeLa cells did lead to a partial decrease in Rheb-GAP activity when compared to controls. Accordingly, shRNA-mediated knockdown of TBC1D7 in serum-starved HeLa cells led to an activation of mTORC1 signalling (as measured by the phosphorylation of S6K, S6 and 4E-BP1). Moreover, the increased phosphorylation of S6K after TBC1D7 knockdown was inhibited by siRNA knockdown of Rheb, confirming that the TBC-TSC complex acts through this protein to augment mTORC1 signalling.
The authors then conducted experiments to see if TBC1D7 plays a role in processes known to be regulated by mTORC1. Following amino acid withdrawal, it could be seen that there was a delay in the initiation of autophagy after shRNA knockdown of TBC1D7 in HeLa cells. There was also a significant increase in HeLa cell size after TBC1D7 knockdown (when compared to siRNA controls). In summary, all these findings support that TBC1D7 is a component of this well-established signalling pathway. Consequently, could mutations in TBC1D7 account for ~15% of patients that are clinically diagnosed with TSC, but have no identifiable mutations in TSC1/TSC2? Dibble et al. exon sequenced the TBC1D7 gene from 12 such patients and found no mutations of significance.
Finally, the TBC domain of many proteins appears to have GAP activity towards specific Rab proteins. However, the function of TBC1D7 is still unclear and it would be interesting to see if it had a similar role. This is of particular relevance to BHD syndrome as Nookala et al. (2012) recently suggested that FLCN may act as a Rab GEF (as discussed in this previous blog post).
- Dibble CC, Elis W, Menon S, Qin W, Klekota J, Asara JM, Finan PM, Kwiatkowski DJ, Murphy LO, & Manning BD (2012). TBC1D7 Is a Third Subunit of the TSC1-TSC2 Complex Upstream of mTORC1. Molecular cell, 47 (4), 535-46 PMID:22795129
- Nookala RK, Langemeyer L, Pacitto A, Ochoa-Montano B, Donaldson JC, Blaszczyk BK, Chirgadze DY, Barr FA, Bazan JF, Blundell TL (2012). Crystal structure of folliculin reveals a hidDENN function in genetically inherited renal cancer Open Biol. DOI: 10.1098/rsob.120071
www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.