DISCUSSION

We herein show for the first time that a variant of the classical bipartite NLS, in which two histidines are separated by a 10-amino-acid spacer in the juxtamembrane region, is necessary for the nuclear translocation of the C-terminal fragment of c-Met. We also demonstrate that this variant is a pH-dependent NLS, providing what we believe to be the first example of such an NLS.

Regarding pH-dependent changes in the subcellular localization of proteins, the p26 protein was shown to undergo nuclear-cytoplasmic shuttling during aerobic-anoxic transition in embryos of the brine shrimp, Artemia franciscana (Clegg et al., 1995). However, this previous study did not examine the amino acid sequences that might be responsible for this phenomenon. A recent study found that von Hippel-Lindau (VHL) tumor suppressor protein is confined to the nucleoli when the extracellular pH drops below normal physiological conditions (Mekhail et al., 2004). However, this prior study did not clearly elucidate the responsible motif or the underlying mechanism for the sub-nuclear confinement of VHL in an acidic environment. To the best of our knowledge, the present study is the first to provide an example of an NLS that shows pH dependency.

The classical bipartite NLSs consists of two stretches of basic amino acid residues normally separated by a 10- to 12-amino-acid linker, as seen in nucleoplasmin (Robbins et al.,1991; Dingwall et al., 1982). Numerous bipartite NLSs have been found in various proteins, as shown in Table 1. Some optimal consensus sequences have been proposed, including (K/R) (K/R) X10-12 (K/R) (Robbins et al., 1991), KRX10-12KRRK (Fontes et al., 2003) and KRX10-12 K (K/R) (Kosugi et al., 2009). Studies on classical bipartite NLS recognitions and interactions have shown that the upstream basic residues bind to the minor groove on importin α, while a downstream monopartite-like sequence combines with the major binding pocket on importin α (Kosugi et al., 2009, Conti et al., 1998 and 2000; Fontes et al., 2000). The latter interaction occurs via salt bridges and H-bonding interactions between positive charges on the NLS and negative charges in the binding pockets of importin α. With two separate binding pockets, importin α can accommodate the 10-residue spacers linking the two basic amino acid clusters in classical bipartite NLSs (Robbins et al.,

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Dingwal et al.,1992). The spacer may be longer, but not smaller, the smaller spacers could not reach two binding pockets. Although, we could not directly show the association between cargo and importin α, the necessity of positive charges for the cargo-importin β interaction is consistent with the widely accepted role of basic amino acid residues in the formation of cargo-importin α – importin β ternary comlex.

Regarding the linker amino acids, the number of linker amino acid usually varies from 10-12 residues long between two basic clusters in bipartite cNLS based on historical characterization of nucleoplasmin cNLS and there are no specific contacts with the adaptor proteins. The precise-amino acid sequences of the spacer are found not to be important and certain much longer spacer segment could be tolerated and still promote efficient nuclear targeting (Dingwall et al., 1982 and Robbins et al., 1991). However, it has also been explained that the sequences of the linker amino acid are very important. But, the specific linkers are required to a particular NLS to adopt a precise conformation or geometry to allow the two basic regions of the NLSs to acquire an orientation that facilitates the interaction with two binding pockets on importin α for transport (Robbins et al., 1991). A recent study has revealed that even a significant longer sequence can be functional (Lange et al., 2010). It has been considered that there is the possibility for binding with the linker region with the body of importin α, which may require specific sequence contacts. Bipartite cNLSs with much longer spacers have also been expected in some cellular proteins such as Smad4 (Xiao et al., 2003), topoisomerase II (Kim et al., 2002), though no extensive study was made to date to prove that whether these unconventional longer sequences are true bipartite cNLSs that direct nuclear import in vivo. Moreover, as in Ty1 integrase in the yeast Saccharomyces cerevisiae harbors an unconventional putative bipartite cNLS containing a 29 amino linker as characterized by ‘KKR-29aa-KKR’ (Moore et al., 1998). However, Ty1 with such an unconventionally longer linker sequence exploits the classical nuclear import machinery via importin-α for its nuclear shuttling. Either the substitution of key basic amino acids with alanines on NLS or the deletion or substitution of amino acid within a series of acidic amino acids in the linker region of Ty1 integrase NLSs abolished the nuclear import (McLane et al., 2008), suggesting that the sequences of linker region is key for facilitating interaction with importin α and subsequent nuclear localization of bipartite NLS- bearing

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cargo. Nonetheless, how the Ty1 integrase cNLS interacts with import receptor, the importin α is still not well defined. Additionally, it has been shown that Rrp4, a subunit of exosome harbors classical bipartite NLSs having a 25- amino- acid linker which is both necessary and sufficient for nuclear import via importin α in vivo (Mitchell et al., 1996). Thus, all these evidences support that the function of the linker region within a classical bipartite NLS is sequence specific and the linker of bipartite NLS and the flanking regions can differentially exploit the surface of importin α and enhance the interaction between NLSs and adaptor protein (Marfori et al., 2011).

Classical nuclear import pathway uses the ternary complex of importin α-importin β1-NLS-containing cargo for nuclear traffic. However, some NLS-bearing cargo proteins without using an adaptor protein importin α directly bind to importin β1.The cargo proteins that are recognized by importin β1 are considerably different from each other.

Nevertheless, there are some previous reports that some NLS-bearing proteins are imported to the nucleus by direct binding with importin β without the involvement of importin α as in Rex protein of HTLV-1(Palmeri et al., 1999; Truant et al., 1999), Smad 3 (Xio et al., 2000), ribosomal protein L23a (Jackel and Gorlich et al., 1998) and histones (Muhlhausser et al., 2001).The specificity for binding of importin β with cargo protein still not well understood.

The putative novel bipartite NLS reported herein differs from the classical bipartite NLSs by having critical histidine residues rather than lysines or arginines. Our results clearly show that these histidine residues are crucial for the nuclear translocation and pH dependency of this NLS. However, the positivity of these individual histidine residues are weaker than those of the two or three lysine/arginine residues commonly present in classical bipartite NLSs. The positivity of histidine residues was expected to be increased with the decrease of intracellular pH either by nigericin treatment or by low pH buffer at the physiological pH range and it might increase the binding potential of the cargo with the carrier proteins via electrostatic interaction forming the trimer complex. In some previous reports, it has been defined that the linker sequences of an NLS, particularly hydrophobic amino acids, are involved in importin-cargo binding (Lange et al., 2010; Fontes et al., 2003 and Jang et al., 2012). But the substitution or deletion of hydrophobic amino acids (1069VVI1071 and 1076LIV1078) in the H1068-H1079 linker of c-Met did not abolish the

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nuclear import (9A, 9C) indicating that only the histidine residues might provide interacting sites for the cargo-importin binding and is sufficient for the association. In addition, this experiment strongly suggests a specific role for the histidines and not a general fold or domain for this region of the protein. The mode of regulatory mechanism or nuclear import pathway of c-Met is still unclear and it requires the further extensive study.

Most of our experiments were performed on both the F-3 fragment and the Jxtm1 fragment (Figure 10D, 11A-D), which is similar to the ~60 kDa C-terminal fragment of c-Met in terms of its molecular size and intramolecular location. Even though, being the smaller size of F-3 fragment, the diffusion probability of F-3 through the NPC cannot be excluded. However, we consistently obtained similar results with the F-3 fragment and Juxtm1. In addition, we found that nigericin treatment up-regulated the endogenous nuclear c-Met fragment in HeLa cells (Figure 13). We are therefore convinced that this pH-dependent NLS exists and functions in the nuclear translocation of the c-Met fragment.

Regarding the opposing signal [i.e., the nuclear export signal (NES) of the c-Met fragment], we found a conserved classical leucine-rich NES (LR-NES) located between residues 1054-1063 of the protein (Figure 14). As expected, alanine substitution of key leucines in the putative NES profoundly enhanced the nuclear localization of a GFP-Met fusion protein (Figure 15A and B), indicating that these leucine residues are critical for the nuclear export of the protein. Moreover, consistent with the mechanisms reported for other LR-NESs (Fornerod et al., 1997 and Fukuda et al.,1997) leptomycin B treatment caused the protein level increased and retained inside the nucleus, indicating that the export activity was exportin-1/CRM1-dependent (Figure 15C and D). Additionally, ATP depletion with 2-deoxyglucose and NaN3 significantly enhanced the nuclear localization of the c-Met fragment, indicating that its nuclear exit of C-terminal fragment is an energy-dependent process (Figure 15E). Therefore, the shuttling of the C-terminal fragment of c-Met between the cytosol and nucleus appears to be due to the presence of both an NLS and an NES in the juxtamembrane region.

What would be the biological relevance of the pH-dependent nuclear accumulation of c-Met fragment? In the first place, the functional role and biological

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relevance of nuclear c-Met is not well understood. However, some authors suggested that C-terminal fragment of nuclear c-Met may play a substantial role as a transcription factor in enhancing the c-Met signaling pathway by activating certain genes which may be associated with either cell proliferation (Rodriguez et al., 2007) or cell migration and motility which leads to invasive phenomenon in MDA-MB231 breast carcinoma cells (Matteucci et al.,2009). pH-dependency of nuclear translocation of c-terminal fragment of c-Met gives us an insight that it may confer proliferative advantage and/or migratory capacity for cancer cells in conditions when cytosolic pH is lowered; that is, hypoxia and/or high lactic acid condition which are frequently encountered in cancers.

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