Previous studies in mouse cell lines have suggested that FLCN, FNIP2 and AMPK are involved in N-Nitroso-N-methylurea (MNU)-induced apoptosis (Lim et al., 2011 – described here). Using a human cell line, further work from the same group has now shown that FNIP2 appears to be stabilised by MNU treatment, and that this stability is modulated by FLCN and AMPK (Sano et al., 2012).
Sano et al. used a HeLa MR cell line which lacks the enzyme MGMT that repairs MNU-induced DNA lesions, and confirmed that FNIP2 is involved in the induction of apoptosis following MNU exposure (using flow cytometry with miRNA-knockdown). Western blot analysis went on to show that FNIP2 protein levels gradually increased after MNU treatment, while those of FLCN and AMPKα remained unchanged. However, qRT-PCR showed no change in the levels of FNIP2 mRNA after MNU treatment, indicating that the observed increase in FNIP2 protein levels was not due to transcriptional effects. Using HeLa MR cells which expressed FLAG-tagged FNIP2, it could be seen that the proteosome inhibitor MG132 led to a significant increase in FNIP2 protein levels, while inhibiting protein synthesis with cyclohexamide led to a significant decrease. In both cases, the levels of FLCN and AMPKα remained constant. Consequently, the authors suggested that FNIP2 may be regulated at the post-transcriptional level following MNU exposure.
As FNIP2 is known to interact with FLCN and AMPK (Hasumi et al., 2008; Takagi et al., 2008; Lim et al., 2011), the authors investigated whether these factors affected the levels of FNIP2. siRNA knockdown of FLCN in HeLa MR cells led to a decrease in FNIP2 protein levels, and treatment with MNU elicited no significant changes. In contrast, siRNA knockdown of AMPKα led to an increase in FNIP2 levels, which increased further after MNU treatment. Together these results suggest that FLCN and AMPK may have opposing effects on the stability of FNIP2. Moreover, the inhibition of AMPK kinase activity with compound C led to an increase in the levels of FNIP2, but no increase was observed after MNU exposure. This result suggests that the kinase activity of AMPK is also linked to the stability of FNIP2.
Finally, immunoprecipitation of FLAG-tagged FNIP2 from HeLa MR cells treated with MNU over 72 hours showed that FLCN interacted with FNIP2 throughout this period. However, it could be seen that the interaction between FNIP2 and AMPKα gradually decreased over the same timescale. Although the exact mechanistic details appear to be quite complex, it is clear that FLCN, FNIP2 and AMPK are associated with MNU-induced apoptosis in vitro.
- Hasumi H, Baba M, Hong SB, Hasumi Y, Huang Y, Yao M, Valera VA, Linehan WM, Schmidt LS. Identification and characterization of a novel folliculin-interacting protein FNIP2. Gene. 2008 May 31;415(1-2):60-7. PMID: 18403135
- Lim TH, Fujikane R, Sano S, Sakagami R, Nakatsu Y, Tsuzuki T, Sekiguchi M, & Hidaka M (2011). Activation of AMP-activated protein kinase by MAPO1 and FLCN induces apoptosis triggered by alkylated base mismatch in DNA. DNA repair PMID: 22209521
- Sano S, Sakagami R, Sekiguchi M, Hidaka M. Stabilization of MAPO1 by specific binding with folliculin and AMP-activated protein kinase in O(6)-methylguanine-induced apoptosis. Biochem Biophys Res Commun. 2012 Nov 29. PMID: 23201403
- Takagi Y, Kobayashi T, Shiono M, Wang L, Piao X, Sun G, Zhang D, Abe M, Hagiwara Y, Takahashi K, Hino O. Interaction of folliculin (Birt-Hogg-Dubé gene product) with a novel Fnip1-like (FnipL/Fnip2) protein. Oncogene. 2008 Sep 11;27(40):5339-47. PMID: 18663353
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