Elsevier

Gene

Volume 415, Issues 1–2, 31 May 2008, Pages 60-67
Gene

Identification and characterization of a novel folliculin-interacting protein FNIP2

https://doi.org/10.1016/j.gene.2008.02.022Get rights and content

Abstract

Birt–Hogg–Dube' syndrome characterized by increased risk for renal neoplasia is caused by germline mutations in the BHD/FLCN gene encoding a novel tumor suppressor protein, folliculin(FLCN), which interacts with FNIP1 and 5′-AMP-activated protein kinase(AMPK). Here we report the identification and characterization of a novel FNIP1 homolog FNIP2 that also interacts with FLCN and AMPK. C-terminally-deleted FLCN mutants, similar to those produced by naturally-occurring germline mutations in BHD patients, were unable to bind FNIP2. These data taken together with our previous results that demonstrated FNIP1 binding to the C-terminus of FLCN suggest that FLCN tumor suppressor function may be facilitated by interactions with both FNIP1 and FNIP2 through its C-terminus. Furthermore, we demonstrate that FNIP1 and FNIP2 are able to form homo- or heteromeric multimers suggesting that they may function independently or cooperatively with FLCN. Differential expression of FNIP1 and FNIP2 transcripts in some normal tissues may indicate tissue specificity for these homologs. Interestingly FNIP1 and FNIP2 were oppositely expressed in human clear cell renal cell carcinoma (RCC), and coordinately expressed in chromophobe RCC and oncocytoma, suggesting their differential function in different histologic variants of RCC.

Introduction

Birt–Hogg–Dubé (BHD) syndrome is an inherited genodermatosis with an increased risk of renal neoplasia (Birt et al., 1977, Toro, 1999, Zbar, 2002) caused by germline mutations in the BHD(FLCN) gene located at chromosome 17p11.2 (Nickerson et al., 2002). Nearly all BHD mutations identified to date, which include frameshift, splice-site or nonsense mutations, are predicted to prematurely truncate the BHD protein, folliculin (FLCN) (Nickerson, 2002, Khoo, 2002, Schmidt, 2005, Leter, 2008). Renal tumors, which develop most frequently in BHD patients, include chromophobe renal cell carcinoma(RCC) and renal oncocytic hybrid tumors (Pavlovich, 2002, Pavlovich, 2005, Murakami, 2007). BHD may function as a tumor suppressor gene since somatic mutations in the remaining wild-type copy of BHD or loss of heterozygosity at chromosome 17p11.2 have been identified in BHD-associated renal tumors (Vocke et al., 2005). Moreover in the Nihon rat model of BHD inactivation, restoration of BHD expression in the rat suppresses renal tumorigenesis (Togashi et al., 2006).

Folliculin is a novel 64-kDa protein without characteristic domains to suggest function (Nickerson et al., 2002). We recently identified FNIP1, a novel folliculin-interacting protein, which also binds to 5′-AMP-activated protein kinase (AMPK) (Baba et al., 2006), an important energy sensor in cells that negatively regulates the master switch for cell growth and proliferation, mammalian target of rapamycin (mTOR) (Inoki et al., 2005). FLCN and FNIP1 phosphorylation levels were affected by AMPK and mTOR activities suggesting a functional relationship with the AMPK-mTOR pathway. Mutations in several other tumor suppressor genes have been shown to result in dysregulation of mTOR signaling leading to the development of other hamartoma syndromes. Here we report the identification of another novel FLCN binding protein FNIP2 (KIAA1450, GenBank accession no. NM_020840) with homology to FNIP1 (49% identity, 74% similarity), which is conserved across species, and also binds to AMPK. Interestingly, FNIP1 and FNIP2 were able to form homo- and heteromeric multimers suggesting a coordinated functional relationship between these proteins. We evaluated expression patterns of FLCN, FNIP1 and FNIP2 in normal human tissues, and compared their expression in sporadic RCC and normal kidney.

Section snippets

FNIP2 identification and bioinformatic analysis

KIAA1450 (GenBank accession no. NM_020840) was identified as a FNIP1 homolog by bioinformatic searching of available sequence databases using BLAST (Altschul et al., 1990). ClustalX (1.8) interface with pairwise gap openings and gap extension penalties set at 10× and 0.2×, respectively, was used for the ClustalW multiple sequence alignment program to prepare multiple alignments of FNIP1, FNIP2 and their homologs. The distances between all pairs of sequences (percent divergence) were calculated

Identification of FNIP2 and evolutionary divergence among FNIP1 and FNIP2 homologs

We have identified a human uncharacterized cDNA, KIAA1450, that encodes a human FNIP1 homolog by bioinformatic searching of available sequence databases using BLAST (Altschul et al., 1990) and designated it FNIP2 (folliculin-interacting protein 2). The KIAA1450 transcript (GenBank accession no. NM_020840) had a 177 bp sequence with a remarkably high GC content upstream of the ATG initiation codon, and we found an in-frame TGA termination codon, which resided in the 5′ end of the GC rich region.

Conclusions

In summary, we have identified a novel FLCN-interacting protein, FNIP2, with homology to FNIP1 that is conserved across species. FNIP2 interacts with the C-terminal half of the folliculin tumor suppressor protein and with an important nutrient- and energy-sensing molecule, AMPK, a negative regulator of mTOR. Importantly we demonstrate that FNIP1 and FNIP2 can form multimers independent of FLCN expression, suggesting that they may function independently or cooperatively with FLCN. Their

Acknowledgements

We acknowledge Kazusa DNA Research Institute as the source of the KIAA1450 clone used in this work. This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. This project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-C0-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Heath

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