14-3-3 suppresses the nuclear localization of threonine 157-phosphorylated p27(Kip1)

p27(Kip1) (p27), a CDK inhibitor, migrates into the nucleus, where it controls cyclin-CDK complex activity for proper cell cycle progression. We report here that the classical bipartite-type basic amino-acid cluster and the two downstream amino acids of the C-terminal region of p27 function as a nuclear localization signal (NLS) for its full nuclear import activity. Importin alpha3 and alpha5, but not alpha1, transported p27 into the nucleus in conjunction with importin beta, as evidenced by an in vitro transport assay. It is known that Akt phosphorylates Thr 157 of p27 and this reduces the nuclear import activity of p27. Using a pull-down experiment, 14-3-3 was identified as the Thr157-phosphorylated p27NLS-binding protein. Although importin alpha5 bound to Thr157-phosphorylated p27NLS, 14-3-3 competed with importin alpha5 for binding to it. Thus, 14-3-3 sequestered phosphorylated p27NLS from importin alpha binding, resulting in cytoplasmic localization of NLS-phosphorylated p27. These findings indicate that 14-3-3 suppresses importin alpha/beta-dependent nuclear localization of Thr157-phosphorylated p27, suggesting implications for cell cycle disorder in Akt-activated cancer cells.     

Bipartite nuclear localization signal of matrin 3 is essential for vertebrate cells

Matrin 3, a nuclear matrix protein has potential (1) to withhold promiscuously edited RNAs within the nucleus in cooperation with p54(nrb) and PSF, (2) to mediate NMDA-induced neuronal death, and (3) to modulate promoter activity of genes proximal to matrix/scaffold attachment region (MAR/SAR). We identified a bipartite nuclear localization signal (NLS) of chicken matrin 3 (cmatr3) at residues 583-602. By expressing green fluorescent protein (GFP) fused to the NLS mutant in chicken DT40 cells, we showed an essential role of the NLS for cell proliferation. Furthermore, we showed that both clusters of basic amino acids and a linker of the bipartite NLS were essential and sufficient for the nuclear import of GFP. Exogenous cmatr3 rescued the HeLa cells where human matrin 3 was suppressed by RNA interference, but cmatr3 containing deletions at either of the basic amino acid clusters or the linker could not.     

Dissection of the karyopherin alpha nuclear localization signal (NLS)-binding groove: functional requirements for NLS binding

Classical protein import, mediated by the binding of a classical nuclear localization signal (NLS) to the NLS receptor, karyopherin/importin alpha, is the most well studied nuclear transport process. Classical NLSs are either monopartite sequences that contain a single cluster of basic amino acids (Lys/Arg) or bipartite sequences that contain two clusters of basic residues separated by an unconserved linker region. We have created mutations in conserved residues in each of the three NLS-binding sites/regions in Saccharomyces cerevisiae karyopherin alpha (SRP1). For each mutant we have analyzed binding to both a monopartite and a bipartite NLS cargo in vitro. We have also expressed each karyopherin alpha mutant in vivo as the only cellular copy of the NLS receptor and examined the impact on cell growth and import of both monopartite and bipartite NLS-containing cargoes. Our results reveal the functional significance of specific residues within karyopherin alpha for NLS cargo binding. A karyopherin alpha variant with a mutation in the major NLS-binding site exhibits decreased binding to both monopartite and bipartite NLS cargoes, and this protein is not functional in vivo. However, we also find that a karyopherin alpha variant with a mutation in the minor NLS-binding site, which shows decreased binding only to bipartite NLS-containing cargoes, is also not functional in vivo. This suggests that the cell is dependent on the function of at least one bipartite NLS cargo that is imported into the nucleus by karyopherin alpha. Our experiments also reveal functional importance for the linker-binding region. This study provides insight into how changes in binding to cellular NLS sequences could impact cellular function. In addition, this work has led to the creation of conditional alleles of karyopherin alpha with well characterized defects in NLS binding that will be useful for identifying and characterizing novel NLS cargoes.     

Importin alpha binds to an unusual bipartite nuclear localization signal in the heterogeneous ribonucleoprotein type I.

The heterogeneous nuclear ribonucleoprotein (hnRNP) type I, a modulator of alternative splicing, localizes in the nucleoplasm of mammalian cells and in a discrete perinucleolar structure. HnRNP I contains a novel type of bipartite nuclear localization signal (NLS) at the N-terminus of the protein that we have previously named nuclear determinant localization type I (NLD-I). Recently, a neural counterpart of hnRNP I has been identified that contains a putative NLS with two strings of basic amino acids separated by a spacer of 30 residues. In the present study we show that the neural hnRNP I NLS is necessary and sufficient for nuclear localization and represents a variant of the novel bipartite NLS present in the NLD-I domain. Furthermore, we demonstrate that the NLD-I is transported into the nucleus by cytoplasmic factor(s) with active transport modality. Binding assays using recombinant importin alpha show an interaction with NLD-I similar to that of SV40 large T antigen NLS. Deletion analysis indicates that both stretches of basic residues are necessary for binding to importin alpha. The above experimental results lead to the conclusion that importin alpha acts as cytoplasmic receptor for proteins characterized by a bipartite NLS signal that extends up to 37 residues.     

Characterization of an unusual importin alpha binding motif in the borna disease virus p10 protein that directs nuclear import.

Nuclear import of many cellular and viral proteins is mediated by short nuclear localization signals (NLS) that are recognized by intracellular receptor proteins belonging to the importin/karyopherin alpha and beta families. The primary structure of NLS is not well defined, but most contain at least three basic amino acids and harbor the relative consensus sequence K(K/R)X(K/R). We have studied the nuclear import of the Borna disease virus p10 protein that lacks a canonical oligobasic NLS. It is shown that the p10 protein exhibits all characteristics of an actively transported molecule in digitonin-permeabilized cells. Import activity was found to reside in the 20 N-terminal p10 amino acids that are devoid of an NLS consensus motif. Unexpectedly, p10-dependent import was blocked by a peptide inhibitor of importin alpha-dependent nuclear translocation, and the transport activity of the p10 N-terminal domain was shown to correlate with the ability to bind to importin alpha. These findings suggest that nuclear import of the Borna disease virus p10 protein occurs through a nonconventional karyophilic signal and highlight that the cellular importin alpha NLS receptor proteins can recognize nuclear targeting signals that substantially deviate from the consensus sequence.     

Nuclear localization of endogenous RGK proteins and modulation of cell shape remodeling by regulated nuclear transport

The members of the RGK small GTP-binding protein family, Kir/Gem, Rad, Rem and Rem2, are multifunctional proteins that regulate voltage-gated calcium channel activity and cell shape remodeling. Calmodulin (CaM) or CaM 14-3-3 are regulators of RGK functions and their association defines the subcellular localization of RGK proteins. Abolition of CaM association results in the accumulation of RGK proteins in the nucleus, whereas 14-3-3 binding maintains them in the cytoplasm. Kir/Gem possesses nuclear localization signals (NLS) that mediate nuclear accumulation through an importin alpha5-dependent pathway (see Mahalakshmi RN, Nagashima K, Ng MY, Inagaki N, Hunziker W, Béguin P. Nuclear transport of Kir/Gem requires specific signals and importin alpha5 and is regulated by Calmodulin and predicted service phosphorylations. Traffic 2007; doi: 10.1111/j.1600-0854.2007.00598.x). Because the extent of nuclear localization depends on the RGK protein and the cell type, the mechanism and regulation of nuclear transport may differ. Here, we extend our analysis to the other RGK members and show that Rem also binds importin alpha5, whereas Rad associates with importins alpha3, alpha5 and beta through three conserved NLS. Predicted phosphorylation of a serine residue within the bipartite NLS affects, as observed for Kir/Gem, nuclear accumulation of Rem, but not that of Rad or Rem2. We also identify an additional regulatory phosphorylation for all RGK proteins that prevents binding of 14-3-3 and thereby interferes with their cytosolic relocalization by 14-3-3. Functionally, nuclear localization of RGK proteins contributes to the suppression of RGK-mediated cell shape remodeling. Importantly, we show that endogenous RGK proteins are localized predominantly in the nucleus of individual cells of the brain cortex 'in situ' as well as in primary hippocampal cells, indicating that transport between the nucleus and their site of action in the cytoplasm (i.e., cytoskeleton, endoplasmic reticulum or plasma membrane) is of physiological relevance for the regulation of RGK protein function.     

Dissection of a nuclear localization signal

The regulated process of protein import into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) that are recognized by protein import receptors. This study seeks to decipher the energetic details of NLS recognition by the receptor importin alpha through quantitative analysis of variant NLSs. The relative importance of each residue in two monopartite NLS sequences was determined using an alanine scanning approach. These measurements yield an energetic definition of a monopartite NLS sequence where a required lysine residue is followed by two other basic residues in the sequence K(K/R)X(K/R). In addition, the energetic contributions of the second basic cluster in a bipartite NLS ( approximately 3 kcal/mol) as well as the energy of inhibition of the importin alpha importin beta-binding domain ( approximately 3 kcal/mol) were also measured. These data allow the generation of an energetic scale of nuclear localization sequences based on a peptide's affinity for the importin alpha-importin beta complex. On this scale, a functional NLS has a binding constant of approximately 10 nm, whereas a nonfunctional NLS has a 100-fold weaker affinity of 1 microm. Further correlation between the current in vitro data and in vivo function will provide the foundation for a comprehensive quantitative model of protein import.     

Expanding the Definition of the Classical Bipartite Nuclear Localization Signal

Nuclear localization signals (NLSs) are amino acid sequences that target cargo proteins into the nucleus. Rigorous characterization of NLS motifs is essential to understanding and predicting pathways for nuclear import. The best‐characterized NLS is the classical NLS (cNLS), which is recognized by the cNLS receptor, importin‐α. cNLSs are conventionally defined as having one (monopartite) or two clusters of basic amino acids separated by a 9‐12 aa linker (bipartite). Motivated by the finding that Ty1 integrase, which contains an unconventional putative bipartite cNLS with a 29 aa linker, exploits the classical nuclear import machinery, we assessed the functional boundaries for linker length within a bipartite cNLS. We confirmed that the integrase cNLS is a bona fide bipartite cNLS, then carried out a systematic analysis of linker length in an obligate bipartite cNLS cargo, which revealed that some linkers longer than conventionally defined can function in nuclear import. Linker function is dependent on the sequence and likely the inherent flexibility of the linker. Subsequently, we interrogated the Saccharomyces cerevisiae proteome to identify cellular proteins containing putative long bipartite cNLSs. We experimentally confirmed that Rrp4 contains a bipartite cNLS with a 25 aa linker. Our studies show that the traditional definition of bipartite cNLSs is too restrictive and linker length can vary depending on amino acid composition.     

A bipartite nuclear localization signal is required for p53 nuclear import regulated by a carboxyl-terminal domain.

Abnormal p53 cellular localization has been considered to be one of the mechanisms that could inactivate p53 function. To understand the regulation of p53 cellular trafficking, we have previously identified two p53 domains involved in its localization. A basic domain, Lys(305)-Arg(306), is required for p53 nuclear import, and a carboxyl-terminal domain, namely the cytoplasmic sequestration domain (CSD) from residues 326-355, could block the nuclear import of Lys(305) or Arg(306) mutated p53. To characterize further the function of these two domains, we demonstrate in this report that the previously described major nuclear localization signal works together with Lys(305)-Arg(306) to form a bipartite and functional nuclear localization sequence (NLS) for p53 nuclear import. The CSD could block the binding of p53 to the NLS receptor, importin alpha, and reduce the efficiency of p53 nuclear import in MCF-7, H1299, and Saos-2 cells. The blocking effect of the CSD is not due to the enhancement of nuclear export or oligomerization of the p53. These results indicate that the CSD can regulate p53 nuclear import by controlling access of the NLS to importin alpha binding.     

Characterization of the auto-inhibitory sequence within the N-terminal domain of importin alpha

Protein cargoes that contain a classic nuclear localization signal (NLS) are transported into the nucleus through binding to a heterodimeric receptor comprised of importin/karyopherin alpha and beta. An evolutionarily conserved auto-inhibitory sequence within the N-terminal importin beta binding (IBB) domain of importin alpha regulates NLS-cargo binding to the NLS binding pocket on importin alpha. In this study, we have used site-directed mutagenesis coupled with in vitro binding assays and in vivo analyses to investigate the intramolecular interaction of the N-terminal IBB domain and the NLS binding pocket of Saccharomyces cerevisiae importin alpha, Srp1p. We find that mutations within the IBB domain that decrease the binding affinity of the auto-inhibitory sequence for the NLS binding pocket impact importin alpha function in vivo. In addition, the severity of the in vivo phenotype is directly correlated to the reduction of auto-inhibition measured in vitro, suggesting that the in vivo phenotypes are directly related to the loss of auto-inhibitory function. We exploit a conditional auto-inhibitory mutant, srp1-55, to study the in vivo functional overlap between the N-terminal IBB domain of importin alpha and other factors implicated in NLS-cargo release, Cse1p and Nup2p. We propose that the N-terminal IBB domain of importin alpha and Cse1p function together in NLS-cargo release, whereas Nup2p contributes to cargo release/importin alpha recycling through a distinct mechanism.