Developmental Cell Essay Keratins Are Going Nuclear Ryan P. Hobbs,1,6 Justin T. Jacob,1,6 and Pierre A. Coulombe1,2,3,4,5,* 1Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA 2Department of Biological Chemistry, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA 3Department of Oncology, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA 4Department of Dermatology, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA 5The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD 21205, USA 6Co-first author *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2016.07.022 Previously thought to reside exclusively in the cytoplasm, the cytoskeletal protein keratin 17 (K17) has been recently identified inside the nucleus of tumor epithelial cells with a direct impact on cell proliferation and gene expression. We comment on fundamental questions raised by this new finding and the associated significance. Everything should be made as regulator autoimmune regulator (AIRE) levels of mRNAs that code for inflamma- simple as possible, but not simpler. (Hobbs et al., 2015) or the cell-cycle inhib- tory and immune cytokines in skin and itor p27KIP1 (CDKN1B) (Escobar-Hoyos cervical tumor paradigms. A key observa- —Albert Einstein et al., 2015)? Whereas a number of intri- tion in the study by Hobbs et al. was cate mechanisms could account for this that manipulating K17 expression had a Intermediate filaments (IFs) are 10-nm- finding, a simple explanation is that K17 marked impact on the distribution of the wide fibrous polymers that, alongside would itself be present and function inside AIRE protein inside the nucleus, i.e., microtubules, F-actin, and associated the nucleus, a possibility that had not punctate versus diffuse, in response to proteins, form the cytoskeleton in multi- been considered until recently. The two relevant stimuli. Escobar-Hoyos et al. (Es- cellular organisms. Remarkably, IFs are aforementioned studies (Hobbs et al., cobar-Hoyos et al., 2015) found K17 to formed by a large and diverse group of 2015; Escobar-Hoyos et al., 2015), in occur in the nucleus of cervical tumor >70 proteins (mass range 20–240 kilo- addition to an unbiased screen to iden- epithelial cells, following up on the obser- daltons) that are expressed and regulated tify nucleocytoplasmic shuttling proteins vation of a slower proliferation rate and in a tissue-, differentiation-, and context- (Kumeta et al., 2013), have separately increased incidence of G1 cell-cycle ar- specific fashion (Pan et al., 2013; Schwe- converged in demonstrating the presence rest when K17 expression is silenced via izer et al., 2006). IFs are abundant of endogenous K17 and other keratin pro- RNAi. In all three studies, uncovering the intracellular elements that partake in all teins inside the nucleus of tumor epithe- presence of keratin proteins inside the nu- basic cellular functions, including growth, lial cells. This unexpected finding raises cleus of cultured tumor cells necessitated death, and virtually everything in between, several questions and calls for new ideas the use of a potent inhibitor of CRM1/Ex- and are necessary to maintain cellular regarding the rationale underlying the portin 1-mediated nuclear export, namely integrity and function in the face of me- context-dependent regulation and signifi- Leptomycin B (LMB), so as to ‘‘trap’’ the chanical and other forms of stress (Pallari cance of K17 and other IF proteins for protein of interest inside the nucleus and and Eriksson, 2006; Pan et al., 2013; cells, tissues, and organs, in both health thus facilitate its detection. This same Toivola et al., 2010). Genetically deter- and disease. strategy had been used before to un- mined mutations in IF proteins account The latest developments regarding nu- cover the presence of other cytoskeletal- for >100 diseases to date (Omary et al., clear keratins are as follows. Kumeta associated proteins, e.g., LPP (lipoma- 2004; Szevereneyi et al., 2008; see the et al. (Kumeta et al., 2013) identified preferred partner; Petit et al., 2000), Zyxin Human Intermediate Filament Database several cytoskeletal proteins, including (Nix et al., 2001), ZO-2 (zona occludens at http://www.interfil.org for an up-to- the actin-binding protein a-actinin, the protein 2; Islas et al., 2002) and KEAP1 date account), rendering their study versatile cellular cytolinker plectin, and (Velichkova and Hasson, 2005), inside immensely relevant to medicine. select keratins (e.g., K7, K8, K17, and the nucleus. No such inhibitory treatment With the notable exception of the K18), in the context of a screen for nuclear is needed, however, to detect nuclear K17 nuclear lamins, all other IFs (n = 68) matrix components that dynamically in BT-20-cultured cells (Figure 1A) and in are believed to reside and function exclu- shuttle in and out of the nucleus in biopsy samples of human BCC skin tu- sively in the cytoplasm. With this in mind, cultured HeLa cells. Hobbs et al. (Hobbs mors by microscopy (Hobbs et al., 2015) how could one account for the puzzling et al., 2015) found K17 to occur in the (Figure 1B), or to biochemically detect observation, independently made by two nucleus of human and mouse tumor the presence of K17 in subcellular frac- laboratories, that the type I IF protein ker- epithelia as part of a quest to decipher tions enriched for nuclear proteins (Esco- atin 17 (K17) impacts the nuclear localiza- the mechanisms underlying the ability of bar-Hoyos et al., 2015; Hobbs et al., 2015; tion and function of the transcriptional this keratin to regulate the induction and Kumeta et al., 2013), which altogether Developmental Cell 38, August 8, 2016 ª 2016 Elsevier Inc. 227 Developmental Cell Essay A D Human epithelial tumor cell line Head Rod Tail BT-20 (breast) HeLa (cervix) A431 (vulva) 1A 1B 2A 2B Type I K17/DAPI K9 K10 B K12 K13 K14 K15 Veh K16 K17 B K18 B K19 B K20 K23 M B K24 K25 B K26 B K27 K28 K31 B K32 B B LMB K33a K33b K34 B K35 B B K36 B K37 K38 K39 B K40 B Human BCC biopsy Type II K1 K17/DAPI K2 K3 K4 B M K5 K6a B K6b B K6c B K7 B K8 K71 B K72 B K73 K74 B K75 B K76 B K77 B K78 B K79 K80 B K81 K82 K83 C 399 400 K84 B K17 (Homo sapiens) -EIATYRRLLEGEDAHLTQYE LT K-KEPVTTRQVRTI-I K85 K86 K17 (Pan troglodytes) -EIATYRRLLEGEDAHLTQYK-KEPVTTRQVRTI- K17 (Mus musculus) NLS -EIATYRRLLEGEDAHLTQYKPKEPVTTRQVRTI- Type III K17 (Xenopus laevis) -EIATYRRLLEGEDAHLSQSQKDGSRTTVQVRTI- Desmin B GFAP B B K17 (Danio rerio) -EIAEYRRLLDGE-ATSVSTSSSKTSTTRKVVTI- Peripherin B B Conservaon Syncoilin B B -555455555455454434333333355445455- Vimenn B Type IV Head Rod Tail α-Internexin B B NF-H B B NF-M B NF-L B B Nesn Synemin-α Synemin-β 194 197 199 204 206 ###*** Type V K17 (Homo sapiens) Lamin A B M M -ADINGLRRVLDELTLARADLEMQIENLKEEL- Lamin B1 M B K17 (Pan troglodytes) -ADINGLRRVLDELTLARADLEMQIENLKEEL- Lamin B2 B M Lamin C1 B M M B NES K17 (Mus musculus) -ADINGLRRVLDELTLARADLEMQIENLKEEL- Lamin C2 B M M B K17 (Xenopus laevis) -ADINGLRRVLDDLTIARSDLEIQIESLKEEL- Type VI K17 (Danio rerio) -ADISGLRKVLDELTMTRSDLEMQIEGLKEEL- Phakinin/CP49 B Conservaon -5554555455545534535554555355555- Filensin/CP115 Figure 1. Nuclear Localization and Nuclear Export and Import Sequences for K17 and All IF Proteins (A and B) Indirect immunostaining for K17 (green) in representative single-plane confocal micrographs of (A) cultured human epithelial tumor cells derived from three tissue types (breast, BT-20 [left]; cervix, HeLa [middle]; vulva, A431 [right]), treated with 40 nM LMB (bottom row) or vehicle (Veh, 70% methanol) (top row), (legend continued on next page) 228 Developmental Cell 38, August 8, 2016 Developmental Cell Essay indicate that nuclear keratins occur in the non-lamin IF proteins have been shown sequence, a highly conserved helix-ter- natural context of tumor cells in culture to associate with or localize to the nu- minating segment that corresponds to and tissues in situ. cleus in various experimental settings. amino acid residue Glycine 390 or Gluta- The idea of nuclear-localized keratins Early studies found that nuclear elements mate 391 in human K17. Interestingly, may not be so far-fetched. Is it not the (e.g., nuclear envelope, matrix, DNA) therefore, the predicted NLS for K17 case, for instance, that other cytoskel- could physically associate with non-lamin spans the boundary between the a-heli- etal proteins previously thought to reside IF proteins added to cells or nuclear ex- cal, coiled-coil-forming rod domain, and function exclusively in the cytoplasm tracts as purified products in vitro, or after which represents the main driving force (e.g., b-catenin, actin, tubulin, and even subjecting cells or tissues to harsh chal- toward assembly into 10-nm filaments myosin motors) have later on been found lenges (e.g., Bastos et al., 1992; Djabali (Herrmann and Aebi, 2004), and the non- to both occur and fulfill important roles et al., 1991; Georgatos and Blobel, 1987; helical tail domain located at the C termi- in the nucleus (McCrea and Gottardi, Tolstonog et al., 2002; Ward et al., nus of K17 (Figure 1C) (Hobbs et al., 2016; Pellegrini and Budman, 2005; 1984). Nuclear-localized keratin proteins 2015). Such a location for a subcellular Philimonenko et al., 2004)? Certainly, the have also been seen in intact culture cell localization signal within the IF protein notion of IF proteins localizing in the models, e.g., when transiently expressing backbone is very