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.     

Overexpression of parathyroid hormone-related protein promotes cell growth in the rat intestinal cell line IEC-6

The rat intestinal cell line, IEC-6, was used as a model to study effects of parathyroid hormone-related protein (PTHrP) on crypt cell growth. Studies showed that addition of PTHrP analogs (1-34), (67-86), or (107-139) to growth medium did not affect proliferation of cells grown in either high (10% Nu-Serum) or low serum (1% Nu-Serum). However, studies on clonal lines of IEC-6 cells stably transfected with PTHrP cDNA and overexpressing PTHrP showed that increased PTHrP production enhanced cell growth and 3H-thymidine incorporation in high, but not low, serum. Additional studies examined the role of the nuclear localization sequence (NLS) of PTHrP in mediating the growth effect. In three clonal IEC-6 lines transfected with PTHrP cDNA bearing a mutated NLS, the ability of PTHrP to stimulate 3H-thymidine incorporation and cell growth was lost. The results suggest that endogenously produced PTHrP can promote proliferation of IEC-6 cells and that the integrity of the NLS of PTHrP is required for its growth effects.     

Cleavage of the ER-targeting signal sequence of parathyroid hormone-related protein is cell-type-specific and regulated in Cis by its nuclear localization signal

Prepro-parathyroid hormone-related protein (ppPTHrP) has two targeting signals, an N-terminal signal sequence and a nuclear localization signal (NLS). In fact, the protein is not only secreted from the cell but also found in the nucleus and/or nucleolus. In order to understand the function of the PTHrP signal sequence for the dual localization, the signal sequence cleavage of a series of ppPTHrP deletion mutants fused to Escherichia coli leader peptidase was analysed in vitro and in several cell lines. Efficiency of the PTHrP signal sequence cleavage was intrinsically low in the in vitro reconstitution system. In cultured cells, cleavage efficiency of the PTHrP signal sequence varied significantly, being lowest in COS-1 cells, but rising in HeLa, HEK293 and CV-1 cells. However, virtually complete signal sequence cleavage was observed in CHO cells. In addition, the NLS of PTHrP had a negative effect on its own signal sequence cleavage, which could be enhanced by deletion of the spacer sequence between the signal sequence and the NLS. There was a roughly inverse relationship between the signal sequence cleavage and the nuclear localization of PTHrP. Thus, the final destination of PTHrP could be regulated at the ER membrane.     

Nuclear PTHrP targeting regulates PTHrP secretion and enhances LoVo cell growth and survival

Parathyroid hormone-related protein (PTHrP) is expressed by human colon cancer tissue and cell lines; expression correlates with colon carcinoma severity. PTHrP is synthesized as a prepro isoform and contains two targeting sequences — a signal sequence and a nuclear localization signal (NLS). The signal peptide (SP) directs PTHrP to the secretory pathway, where it exerts autocrine/paracrine effects. The NLS directs PTHrP to the nucleus/nucleolus, where it exerts intracrine effects. In this study, we used the human colon cancer cell line LoVo as a model system to study the effects of autocrine/paracrine and intracrine PTHrP action on cell growth and survival, hallmarks of malignant tumor cells. We report that PTHrP increases cell growth and survival, protects cells from serum-starvation-induced apoptosis, and promotes anchorage-independent cell growth via an intracrine pathway. Conversely, autocrine/paracrine PTHrP action decreases cell growth and survival. We also show an inverse relationship between secreted and nuclear PTHrP levels, in that cells overexpressing NLS-deleted PTHrP secrete higher PTHrP levels than those overexpressing the wild-type isoform. Conversely, SP deletion results in higher nuclear PTHrP levels. These observations provide evidence of a link between intracrine PTHrP action and cell growth and survival. Targeting PTHrP production in colon cancer may thus prove therapeutically beneficial.     

Parathyroid hormone-related protein upregulates integrin expression via an intracrine pathway in PC-3 prostate cancer cells

Parathyroid hormone-related protein (PTHrP) is expressed by human prostatic tissue and prostate cancer cell lines, and enhances prostate tumor cell growth both in vivo and in vitro. PTHrP expression also plays a role in the development of bone metastasis, which is a frequent complication in patients with prostate carcinoma. Tumor cell adhesion to extracellular matrix (ECM) components is mediated via integrin subunits, and plays a major role in the invasion and metastasis of tumor cells. We previously showed that PTHrP overexpression increases adhesion of the human prostate cancer cell line PC-3 to the ECM molecules collagen type I, fibronectin, and laminin. Increased adhesion is accompanied by upregulation in the expression of alpha1, alpha5, alpha6, and beta4 integrin subunits. We used the same cell line to study the mechanism via which PTHrP upregulates integrin expression. Clonal PC-3 cells were established overexpressing wild-type PTHrP or PTHrP mutated in the nuclear localization sequence (NLS). Mutation of the NLS negated the effects of PTHrP on alpha1, alpha5, alpha6, and beta4 integrin expression, indicating that these effects are mediated via an intracrine pathway requiring nuclear localization. Expression of the alpha2, alpha3, alphav, and beta1 integrin subunits were comparable in wild-type and NLS-mutated PTHrP transfectants. These findings indicate that PTHrP may play a role in prostate tumor invasion and metastasis by upregulating the expression of specific integrin subunits via an intracrine pathway.     

Parathyroid hormone-related protein enhances PC-3 prostate cancer cell growth via both autocrine/paracrine and intracrine pathways

Parathyroid hormone-related protein (PTHrP) is expressed by human prostatic tissue and prostate cancer cell lines, and positively influences primary prostate tumor growth in vivo. The human prostate cancer cell line PC-3, which expresses functional PTH/PTHrP receptors, was used as a model to study the effects of PTHrP on prostate cancer cell growth. Addition of PTHrP (1-34), (1-86), and (1-139) increased cell number and [3H]thymidine incorporation; these effects were reversed by anti-PTHrP antiserum. This antiserum also decreased endogenous PC-3 cell growth. Clonal PTHrP-overexpressing PC-3 cell lines also showed enhanced cell growth and [3H]thymidine incorporation and were enriched in the G2+M phase of the cell cycle, suggesting an effect of PTHrP on mitosis. Overexpression of PTHrP with the nuclear localization sequence (NLS) deletion partially reversed the growth-stimulatory effects. The growth rate of these cells was midway between that of wild-type PTHrP-overexpressing and control cells, presumably because NLS-mutated PTHrP is still secreted and acts through the cell surface PTH/PTHrP receptor. In contrast to NLS-mutated PTHrP, wild-type protein showed preferential nuclear localization. These results suggest that the proliferative effects of PTHrP in PC-3 cells are mediated via both autocrine/paracrine and intracrine pathways, and that controlling PTHrP production in prostate cancer may be therapeutically beneficial.     

PTH-related protein enhances MCF-7 breast cancer cell adhesion, migration, and invasion via an intracrine pathway

Breast cancer is the most common carcinoma that metastasizes to the bone. Parathyroid hormone-related protein (PTHrP), a known stimulator of osteoclastic bone resorption, is a major mediator of the osteolytic process in breast cancer. PTHrP overexpression increases mitogenesis and decreases apoptosis in the human breast cancer cell line MCF-7. In this study, MCF-7 cells were used as a model system to study the effects of PTHrP on breast cancer cell adhesion, migration, and invasion. Clones of MCF-7 cells were established that overexpress wild-type PTHrP or PTHrP mutated in the nuclear localization sequence (NLS). Wild-type PTHrP-overexpressing cells showed significantly higher laminin adhesion and migration, and Matrigel invasion than empty vector-transfectants or cells overexpressing NLS-mutated PTHrP. Wild-type PTHrP also increased the cell surface expression of the pro-invasive integrins alpha6 and beta4; deletion of the NLS negated these effects. Exogenous PTHrP (1-34), (67-86), (107-139), and (140-173) had no effect on integrin expression, or on cell adhesion, migration, and invasion. These results indicate that PTHrP exerts its effects on cell adhesion, migration, invasion, and integrin expression via an intracrine pathway. PTHrP may play a role in breast cancer metastasis by upregulating proinvasive integrin expression, and controlling PTHrP production in breast cancer may provide therapeutic benefit.     

Overlapping signals for protein degradation and nuclear localization define a role for intrinsic RAG-2 nuclear uptake in dividing cells

Expression of the recombinase proteins RAG-1 and RAG-2 is discordant: while RAG-1 is relatively long lived, RAG-2 is degraded periodically at the G(1)-S transition. Destruction of RAG-2 is mediated by a conserved interval in the recombination-dispensable region. The need for RAG-2 to reaccumulate in the nucleus at each cell division suggested the existence of an intrinsic RAG-2 nuclear localization signal (NLS). RAG-1 or RAG-2, expressed individually, is a nuclear protein. A screen for proteins that bind the recombination-dispensable region of RAG-2 identified the nuclear transport protein Importin 5. Mutation of residues 499 to 508 in RAG-2 abolished Importin 5 binding, nuclear accumulation, and periodic degradation of RAG-2. The Importin 5 binding site overlaps an NLS, defined by mutagenesis. RAG-1 rescued the localization of degradation-defective, RAG-2 NLS mutants; this required an intact RAG-1 NLS. Mutations in RAG-2 that abolish intrinsic nuclear accumulation but spare periodic degradation impaired recombination in cycling cells; induction of quiescence restored recombination to wild-type levels. Recombination defects were correlated with a cell cycle-dependent defect in the ability of RAG-1 to rescue localization of the RAG-2 mutants. These results suggest that the intrinsic RAG-2 NLS functions in the nuclear uptake of RAG-2 following its reexpression in cycling cells.     

Nuclear but not cytosolic phosphoinositide 3-kinase beta has an essential function in cell survival

Class I(A) phosphoinositide 3-kinases (PI3Ks) are heterodimeric enzymes composed of a p85 regulatory and a p110 catalytic subunit that induce the formation of 3-polyphosphoinositides, which mediate cell survival, division, and migration. There are two ubiquitous PI3K isoforms p110α and p110β that have nonredundant functions in embryonic development and cell division. However, whereas p110α concentrates in the cytoplasm, p110β localizes to the nucleus and modulates nuclear processes such as DNA replication and repair. At present, the structural features that determine p110β nuclear localization remain unknown. We describe here that association with the p85β regulatory subunit controls p110β nuclear localization. We identified a nuclear localization signal (NLS) in p110β C2 domain that mediates its nuclear entry, as well as a nuclear export sequence (NES) in p85β. Deletion of p110β induced apoptosis, and complementation with the cytoplasmic C2-NLS p110β mutant was unable to restore cell survival. These studies show that p110β NLS and p85β NES regulate p85β/p110β nuclear localization, supporting the idea that nuclear, but not cytoplasmic, p110β controls cell survival.     

Phosphorylation-dependent control of ZIPK nuclear import is species specific

ZIPK (zipper-interacting protein kinase) is a Ca²⁺-independent protein kinase that promotes myosin phosphorylation in both smooth muscle and non-muscle cells. A recent report attempted to clarify a debate over the subcellular localization of ZIPK in non-muscle cells (Shoval et. al. (2007) Plos Genetics. 3: 1884-1883). A species-specific loss of a key phosphorylation site (T299) in murine (mouse and rat) ZIPK seems to direct it to the nucleus, while the presence of the T299 site in human ZIPK correlates with cytoplasmic localization. T299 is immediately adjacent to a putative nuclear localization sequence (NLS) and may mask its function when phosphorylated, therefore explaining the species-specific dichotomy of intracellular localization. However, despite the murine ZIPK (mZIPK) lacking the T299 residue that is critical for controlling human ZIPK (hZIPK) subcellular localization, mutational analysis showed that this NLS control locus is nonfunctional in the murine context. A constitutively active Rho promoted the cytoplasmic retention of a human ZIPK mutant that would otherwise localize to the nucleus. Endogenous hZIPK showed sensitivity to the nuclear export inhibitor leptomycin B, suggesting a continuous shuttling between cytoplasm and nucleus that is dependent upon T299 dephosphorylation. Thus, the C-terminal domain of human and murine ZIPK demonstrated quite divergent nuclear import and export functionality. We conclude that in the case of ZIPK, studies between the species may not be directly comparable to each other given the gross differences in intracellular localization and movement.     

Nuclear import and export of Venezuelan equine encephalitis virus nonstructural protein 2

Many RNA viruses, which replicate predominantly in the cytoplasm, have nuclear components that contribute to their life cycle or pathogenesis. We investigated the intracellular localization of the multifunctional nonstructural protein 2 (nsP2) in mammalian cells infected with Venezuelan equine encephalitis virus (VEE), an important, naturally emerging zoonotic alphavirus. VEE nsP2 localizes to both the cytoplasm and the nucleus of mammalian cells in the context of infection and also when expressed alone. Through the analysis of a series of enhanced green fluorescent protein fusions, a segment of nsP2 that completely localizes to the nucleus of mammalian cells was identified. Within this region, mutation of the putative nuclear localization signal (NLS) PGKMV diminished, but did not obliterate, the ability of the protein to localize to the nucleus, suggesting that this sequence contributes to the nuclear localization of VEE nsP2. Furthermore, VEE nsP2 specifically interacted with the nuclear import protein karyopherin-alpha1 but not with karyopherin-alpha2, -3, or -4, suggesting that karyopherin-alpha1 transports nsP2 to the nucleus during infection. Additionally, a novel nuclear export signal (NES) was identified, which included residues L526 and L528 of VEE nsP2. Leptomycin B treatment resulted in nuclear accumulation of nsP2, demonstrating that nuclear export of nsP2 is mediated via the CRM1 nuclear export pathway. Disruption of either the NLS or the NES in nsP2 compromised essential viral functions. Taken together, these results establish the bidirectional transport of nsP2 across the nuclear membrane, suggesting that a critical function of nsP2 during infection involves its shuttling between the cytoplasm and the nucleus.     

Multiple mechanisms regulate subcellular localization of human CDC6

CDC6 is a protein essential for DNA replication, the expression and abundance of which are cell cycle-regulated in Saccharomyces cerevisiae. We have demonstrated previously that the subcellular localization of the human CDC6 homolog, HsCDC6, is cell cycle-dependent: nuclear during G(1) phase and cytoplasmic during S phase. Here we demonstrate that endogenous HsCDC6 is phosphorylated during the G(1)/S transition. The N-terminal region contains putative cyclin-dependent kinase phosphorylation sites adjoining nuclear localization sequences (NLSs) and a cyclin-docking motif, whereas the C-terminal region contains a nuclear export signal (NES). In addition, we show that the observed regulated subcellular localization depends on phosphorylation status, NLS, and NES. When the four putative substrate sites (serines 45, 54, 74, and 106) for cyclin-dependent kinases are mutated to alanines, the resulting HsCDC6A4 protein is localized predominantly to the nucleus. This localization depends upon two functional NLSs, because expression of HsCDC6 containing mutations in the two putative NLSs results in predominantly cytoplasmic distribution. Furthermore, mutation of the four serines to phosphate-mimicking aspartates (HsCDC6D4) results in strictly cytoplasmic localization. This cytoplasmic localization depends upon the C-terminal NES. Together these results demonstrate that HsCDC6 is phosphorylated at the G(1)/S phase of the cell cycle and that the phosphorylation status determines the subcellular localization.     

Phosphorylation mediates the nuclear targeting of the maize Rab17 protein

The maize abscisic acid-responsive Rab17 protein localizes to the nucleus and cytoplasm in maize cells. In-frame fusion of Rab17 to the reporter protein β-glucuronidase (GUS) directed GUS to the nucleus and cytoplasm in transgenic Arabidopsis thaliana and in transiently transformed onion cells. Analysis of chimeric constructs identified one region between amino acid positions 66–96, which was necessary for targeting GUS to the nucleus. This region contains a serine cluster followed by a putative consensus site for protein kinase CK2 phosphorylation, and a stretch of basic amino acids resembling the simian virus 40 large T antigen-type nuclear localization signal (NLS). Mutation of two basic amino acids in the putative NLS had a weak effect on nuclear targeting in the onion cell system and did not modify the percentage of nuclear fusion protein in the Arabidopsis cells. The mutation of three amino acids in the consensus site for CK2 recognition resulted in the absence of in vitro phosphorylated forms of Rab17 and in a strong decrease of GUS enzymatic activity in isolated nuclei of transgenic Arabidopsis. These results suggest that phosphorylation of Rab17 by protein kinase CK2 is the relevant step for its nuclear location, either by facilitating binding to specific proteins or as a direct part of the nuclear targeting apparatus.     

The Intracellular Mobility of Nuclear Import Receptors and NLS Cargoes

We have investigated classical nuclear localization sequence (NLS) mediated protein trafficking by measuring biomolecular dynamics within living cells using two-photon fluorescence correlation spectroscopy. By directly observing the behavior of specific molecules in their native cellular environment, it is possible to uncover functional details that are not apparent from traditional biochemical investigations or functional assays. We show that the intracellular mobility of NLS cargoes and their import receptor proteins, karyopherin-α and karyopherin-β, can be robustly measured and that quantitative comparison of intracellular diffusion coefficients provides new insights into nuclear transport mechanisms. Import cargo complexes are assembled throughout the cytoplasm, and their diffusion is slower than predicted by molecular weight due to specific interactions. Analysis of NLS cargo diffusion in the cytoplasm indicates that these interactions are likely disrupted by NLS cargo binding. Our results suggest that delivery of import receptors and NLS cargoes to nuclear pores may complement selective translocation through the pores as a functional mechanism for regulating transport of proteins into the nucleus.     

Two nuclear localization signals required for transport from the cytosol to the nucleus of externally added FGF-1 translocated into cells

Externally added FGF-1 is transported into the nucleus of cells. It was earlier shown that FGF-1 contains an N-terminal nuclear localization signal (NLS) implicated in the stimulation of DNA synthesis. We here provide evidence that FGF-1 contains a second putative NLS (NLS2), which is located near the C-terminus. It is a bipartite NLS consisting of two clusters of lysines separated by a spacer of 10 amino acids. A fusion protein of GFP and the bipartite NLS was more efficiently transported into the nucleus than GFP alone, indicating that it can act as an NLS in the living cell. FGF-1 mutated in the N-terminal NLS (NLS1) or in the first cluster of the bipartite NLS2 bound to heparin and FGF receptors and activated downstream signaling similarly to the wild-type growth factor. Mutations in the second cluster of NLS2 resulted in impaired interaction with heparin and reduced stability. When radiolabeled FGF-1 with mutated NLS1 or the first lysine cluster of NLS2 was added to NIH/3T3 cells, it was translocated into the cytosol, but not transported efficiently to the nucleus. Phosphorylation of FGF-1 occurs normally in the nucleus, and while wild-type FGF-1 was phosphorylated after addition to cells, the NLS mutants were not. It therefore appears that both NLS1 and NLS2 are important for efficient transport of FGF-1 to the nucleus. Stimulation of DNA synthesis by FGF-1 with mutations in both NLSs was reduced considerably indicating that efficient transport to the nucleus may be involved in the stimulation of DNA synthesis.