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.     

FGF-1 and FGF-2 require the cytosolic chaperone Hsp90 for translocation into the cytosol and the cell nucleus

Similarly to many protein toxins, the growth factors fibroblast growth factor 1 (FGF-1) and FGF-2 translocate from endosomes into the cytosol. It was recently found that certain toxins are dependent on cytosolic Hsp90 for efficient translocation across the endosomal membrane. We therefore investigated the requirement for Hsp90 in FGF translocation. We found that low concentrations of the specific Hsp90 inhibitors, geldanamycin and radicicol, completely blocked the translocation of FGF-1 and FGF-2 to the cytosol and the nucleus. The drugs did not interfere with the initial binding of FGF-1 to the growth factor receptors at the cell-surface or with the subsequent internalization of the growth factors into endosomes. The activation of known signaling cascades downstream of the growth factor receptors was also not affected by the drugs. The data indicate that the drugs block translocation from endosomes to the cytosol implying that Hsp90 is required for translocation of FGF-1 and FGF-2 across the endosomal membrane.     

Cyclin-dependent protein kinase 2 activity is required for mitochondrial translocation of Bax and disruption of mitochondrial transmembrane potential during etoposide-induced apoptosis

Previous studies have suggested that upregulation of Cyclin A-dependent protein kinase 2 (Cdk2) activity is an essential event in apoptotic progression and the mitochondrial permeability transition in human cancer cells. Here, we show that upregulated Cyclin A/Cdk2 activity precedes the proteolytic cleavage of PARP and is correlated with the mitochondrial translocation of Bax and the loss of mitochondrial transmembrane potential (Δψm) during etoposide-induced apoptosis in human cervical adenocarcinoma (HeLa) cells. Etoposide-induced apoptotic cell death is efficiently prevented in cells that overexpress a dominant negative mutant of Cdk2 (Cdk2-dn) or p21^WAF1/CIP1, a specific Cdk inhibitor. Conversely, apoptotic cell death is promoted in Cyclin A-expressing cells. Disruption of the mitochondrial transmembrane potential in etoposide-induced cells is prevented in cells that overexpress Cdk2-dn or p21^WAF1/CIP1, while this transition is prominently promoted in Cyclin A-expressing cells. We screened for mitochondrial Cdk2 targets in the etoposide-induced cells and found that the mitochondrial level of Bax is elevated by more than three fold in etoposide-treated cells and this elevation is effectively prevented in cells expressing Cdk2-dn under the same conditions. Thus, we suggest that Cdk2 activity is involved in the mitochondrial translocation of Bax, which plays an important role in the mitochondrial membrane permeability transition during apoptotic progression.     

FGF-16 is required for embryonic heart development

Fibroblast growth factor 16 (FGF-16) expression has previously been detected in mouse heart at mid-gestation in the endocardium and epicardium, suggesting a role in embryonic heart development. More specifically, exogenously applied FGF-16 has been shown to stimulate growth of embryonic myocardial cells in tissue explants. We have generated mice lacking FGF-16 by targeting the Fgf16 locus on the X chromosome. Elimination of Fgf16 expression resulted in embryonic death as early as day 11.5 (E11.5). External abnormalities, including hemorrhage in the heart and ventral body region as well as facial defects, began to appear in null embryos from E11.5. Morphological analysis of FGF-16 null hearts revealed cardiac defects including chamber dilation, thinning of the atrial and ventricular walls, and poor trabeculation, which were visible at E10.5 and more pronounced at E11.5. These findings indicate FGF-16 is required for embryonic heart development in mid-gestation through its positive effect on myocardial growth.     

The transmembrane nucleoporin NDC1 is required for targeting of ALADIN to nuclear pore complexes

NDC1 is a transmembrane nucleoporin that is required for NPC assembly and nucleocytoplasmic transport. We show here that NDC1 directly interacts with the nucleoporin ALADIN, mutations of which are responsible for triple-A syndrome, and that this interaction is required for targeting of ALADIN to nuclear pore complexes (NPCs). Furthermore, we show that NDC1 is required for selective nuclear import. Our findings suggest that NDC1-mediated localization of ALADIN to NPCs is essential for selective nuclear protein import, and that abrogation of the interaction between ALADIN and NDC1 may be important for the development of triple-A syndrome.     

ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria

Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patient-derived mycobacteria species were found to translocate to the cytosol, while non-pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX-1 (type VII) secretion system into the non-virulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C-terminus of the early-secreted antigen ESAT-6. The C-terminal truncation of ESAT-6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence.     

TSC1 binding to lysosomal PIPs is required for TSC complex translocation and mTORC1 regulation

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Collapse of the inner mitochondrial transmembrane potential is not required for apoptosis of HL60 cells.

Apoptotic cell death involves a series of morphological and biochemical changes orchestrated by activated proteases belonging to the caspase family. Recent studies have suggested that the activation of this process of execution is dependent upon events associated with the loss of mitochondrial inner transmembrane potential (Deltapsi(m)), as a consequence of the formation of the permeability transition (PT) pore. This has led to the proposal that mitochondrial depolarization represents a central irreversible checkpoint in the apoptotic program. Here, we present evidence that HL-60 cells undergo apoptosis in response to the cytotoxic insults of actinomycin-D, etoposide, and staurosporine without showing significant changes in Deltapsi(m). Instead, the loss of Deltapsi(m) could be detected only later in the cell death pathway. In addition, the uncoupling agent CCCP produced an early mitochondrial depolarization in HL-60s but these cells showed few signs of apoptosis up to 8 h after the insult. Furthermore, examination of these cells in response to staurosporine revealed the release of mitochondrial cytochrome c into the cytosol over time, corresponding to caspase activation irrespective of mitochondrial depolarization. In summary, our data suggest that the collapse of Deltapsi(m) as a consequence of PT is not a universal early marker for apoptosis and, moreover, it is not part of the central apoptotic machinery.     

Bafilomycin A1-sensitive pathway is required for the maturation of cystic fibrosis transmembrane conductance regulator

Cystic fibrosis (CF) is the most common lethal genetic disease in Caucasians caused by the trafficking defects of CF transmembrane conductance regulator (CFTR), which is a cAMP-dependent Cl- channel at the plasma membrane. The trafficking pathway of CFTR is thought to be non-conventional because CFTR maturation is inhibited by the dysfunction of syntaxin 13, which is involved in protein recycling via endosomal pathway. In this study, to clarify whether the endosomal trafficking is required for CFTR maturation, we utilized a specific vacuolar H+-ATPase inhibitor, bafilomycin A1 (BafA1), which inhibits the protein trafficking from early endosome. Our data showed that low concentration of BafA1 (50 nM) decreased the expression of mature CFTR but induced the accumulation of immature CFTR in the juxta-nuclear region containing an early endosome marker. Pulse-chase analysis showed that BafA1 inhibited the maturation of CFTR, but it slightly stabilized immature CFTR. These results indicate that BafA1-sensitive pathway is required for CFTR maturation and emphasize that endosomal trafficking pathway might be involved in the maturation of CFTR.     

A transmembrane leucine zipper is required for activation of the dimeric receptor tyrosine kinase DDR1

Receptor tyrosine kinases of the discoidin domain family, DDR1 and DDR2, are activated by different types of collagen and play important roles in cell adhesion, migration, proliferation, and matrix remodeling. In a previous study, we found that collagen binding by the discoidin domain receptors (DDRs) requires dimerization of their extracellular domains (Leitinger, B. (2003) J. Biol. Chem. 278, 16761-16769), indicating that the paradigm of ligand-induced receptor dimerization may not apply to the DDRs. Using chemical cross-linking and co-immunoprecipitation of differently tagged DDRs, we now show that the DDRs form ligand-independent dimers in the biosynthetic pathway and on the cell surface. We further show that both the extracellular and the cytoplasmic domains are individually dispensable for DDR1 dimerization. The DDR1 transmembrane domain contains two putative dimerization motifs, a leucine zipper and a GXXXG motif. Mutations disrupting the leucine zipper strongly impaired collagen-induced transmembrane signaling, although the mutant DDR1 proteins were still able to dimerize, whereas mutation of the GXXXG motif had no effect. A bacterial reporter assay (named TOXCAT) showed that the DDR1 transmembrane domain has a strong potential for self-association in a biological membrane and that this interaction occurs via the leucine zipper and not the GXXXG motif. Our results demonstrate that the DDRs exist as stable dimers in the absence of ligand and that receptor activation requires specific interactions made by the transmembrane leucine zipper.     

The interaction of Akt with APPL1 is required for insulin-stimulated Glut4 translocation

APPL1 (adaptor protein containing PH domain, PTB domain, and leucine zipper motif 1) is an Akt/protein kinase B-binding protein involved in signal transduction and membrane trafficking pathways for various receptors, including receptor tyrosine kinases. Here, we establish a role for APPL1 in insulin signaling in which we demonstrate its interaction with Akt2 by co-immunoprecipitation and pulldown assays. In primary rat adipocytes and skeletal muscle, APPL1 and Akt2 formed a complex that was dissociated upon insulin stimulation in both tissues. To investigate possible APPL1 function in adipocytes, we analyzed Akt phosphorylation, 2-deoxyglucose uptake, and Glut4 translocation by immunofluorescence following APPL1 knockdown by small interfering and short hairpin RNAs. We show that APPL1 knockdown suppressed Akt phosphorylation, glucose uptake, and Glut4 translocation. We also tested the effect in 3T3-L1 adipocytes of expressing full-length APPL1 or an N- or a C-terminal APPL1 construct. Interestingly, expression of full-length APPL1 and its N terminus suppressed insulin-stimulated 2-deoxyglucose uptake and Glut4 translocation to roughly the same extent (40-60%). We confirmed by cellular fractionation that Glut4 translocation was substantially blocked in 3T3-L1 adipocytes transfected with full-length APPL1. By cellular fractionation, APPL1 was localized mainly in the cytosol, and it showed a small degree of re-localization to the light microsomes and nucleus in response to insulin. By immunofluorescence, we also show that APPL1 partially co-localized with Glut4. These data suggest that APPL1 plays an important role in insulin-stimulated Glut4 translocation in muscle and adipose tissues and that its N-terminal portion may be critical for APPL1 function.     

Structural requirements of FGF-1 for receptor binding and translocation into cells

FGF-1 binds to and activates specific transmembrane receptors (FGFRs) and is subsequently internalized and translocated to the interior of the cell. To elucidate the role of the receptor in the translocation process, we studied the effects of the elimination of distinct sites of the ligand-receptor interaction. On the basis of the structure of the FGF-1-FGFR1 complex, we substituted four key amino acid residues of FGF-1 from the FGF-receptor binding site with alanines, constructing four point mutants and one double mutant. We determined by in vivo assays in NIH 3T3 cells the ability of the mutants to bind to specific FGF receptors, to stimulate DNA synthesis, and to activate downstream signaling pathways. We found that correct binding to the receptor is necessary for optimal stimulation of DNA synthesis. All four single mutants became phosphorylated to different extents, indicating that they were translocated to the cytosol/nucleus with varying efficiency. This indicates that despite a low affinity for FGFR, translocation to the cytosol/nucleus can still occur. However, simultaneous substitution in two of the positions led to a total loss of biological activity of the growth factor and prevented its internalization, implying that there is only one strongly receptor-dependent, productive way of translocating FGF-1. We also found that the process of translocation did not correlate with the thermal stability of the protein. Additionally, we observed a clear negative correlation between the stability of the FGF-1 mutants and the efficiency of their phosphorylation, which strongly suggests that protein kinases prefer the unfolded state of the protein substrate.     

Synip phosphorylation is required for insulin-stimulated Glut4 translocation

Previously we identified an unusual potential dual Akt/protein kinase B consensus phosphorylation motif in the protein Synip (RxKxRS(97)xS(99)) with serine 99 as a unique Akt2, but not Akt1 or for Akt3, substrate phosphorylation site. Although we have previously reported that serine 99 to phenylalanine (S99F-Synip) resulted in a constitutive inhibition of insulin-stimulated Glut4 translocation, a recent report indicated that Synip serine 99 to alanine mutant (S99A-Synip) failed to inhibit insulin-stimulated Glut4 translocation [H. Sano, S. Kane, E. Sano, G.E. Lienhard, Synip phosphorylation does not regulate insulin-stimulated GLUT4 translocation, Biochem. Biophys. Res. Commun. 332 (2005) 880-884]. To address this apparent discrepancy, we have now examined the S99A-Synip mutant and find that this mutant behaves essentially identical to S99F-Synip in that overexpression inhibits insulin-stimulated Glut4 translocation and is incapable of undergoing insulin-stimulated Syntaxin4 dissociation. These data are consistent with Synip serine 99 phosphorylation required for insulin-stimulated Glut4 translocation.     

MEI-1/katanin is required for translocation of the meiosis I spindle to the oocyte cortex in C elegans

In most animals, successful segregation of female meiotic chromosomes involves sequential associations of the meiosis I and meiosis II spindles with the cell cortex so that extra chromosomes can be deposited in polar bodies. The resulting reduction in chromosome number is essential to prevent the generation of polyploid embryos after fertilization. Using time-lapse imaging of living Caenorhabditis elegans oocytes containing fluorescently labeled chromosomes or microtubules, we have characterized the movements of meiotic spindles relative to the cell cortex. Spindle assembly initiated several microns from the cortex. After formation of a bipolar structure, the meiosis I spindle translocated to the cortex. When microtubules were partially depleted, translocation of the bivalent chromosomes to the cortex was blocked without affecting cell cycle timing. In oocytes depleted of the microtubule-severing enzyme, MEI-1, spindles moved to the cortex, but association with the cortex was unstable. Unlike translocation of wild-type spindles, movement of MEI-1-depleted spindles was dependent on FZY-1/CDC20, a regulator of the metaphase/anaphase transition. We observed a microtubule and FZY-1/CDC20-dependent circular cytoplasmic streaming in wild-type and mei-1 mutant embryos during meiosis. We propose that, in mei-1 mutant oocytes, this cytoplasmic streaming is sufficient to drive the spindle into the cortex. Cytoplasmic streaming is not the normal spindle translocation mechanism because translocation occurred in the absence of cytoplasmic streaming in embryos depleted of either the orbit/CLASP homolog, CLS-2, or FZY-1. These results indicate a direct role of microtubule severing in translocation of the meiotic spindle to the cortex.     

Ubiquitylation is required for degradation of transmembrane surface proteins in trypanosomes

The surface of Trypanosoma brucei is dominated by glycosyl-phosphatidylinositol (GPI)-anchored proteins, and endocytosis is clathrin dependent. The vast majority of internalized GPI-anchored protein is efficiently recycled, while the processes by which transmembrane domain (TMD) proteins are internalized and sorted are unknown. We demonstrate that internalization of invariant surface glycoprotein (ISG)65, a trypanosome TMD protein, involves ubiquitylation and also requires clathrin. We find a hierarchical requirement for cytoplasmic lysine residues in internalization and turnover, and a single position-specific lysine is sufficient for degradation, surface removal and attachment of oligoubiquitin chains. Ubiquitylation is context dependent as provision of additional lysine residues by C-terminal fusion of neuronal precursor cell-expressed developmentally downregulated protein (NEDD)8 fails to support ubiquitylation. Attachment of NEDD8 leads to degradation by a second ubiquitin-independent pathway. Moreover, degradation of ubiquitylated or NEDDylated substrate takes place in an acidic compartment and is proteosome independent. Significantly, in non-opisthokont lineages, Rsp5p or c-Cbl, the E3 ubiquitin ligases acting on endocytic cargo, are absent but Uba1 class genes are present and are required for cell viability and ISG65 ubiquitylation. Hence, ubiquitylation is an evolutionarily conserved mechanism for internalization of surface proteins, but aspects of the machinery differ substantially between the major eukaryotic lineages.     

PPARgamma1 attenuates cytosol to membrane translocation of PKCalpha to desensitize monocytes/macrophages

Recently, we provided evidence that PKCalpha depletion in monocytes/macrophages contributes to cellular desensitization during sepsis. We demonstrate that peroxisome proliferator-activated receptor gamma (PPARgamma) agonists dose dependently block PKCalpha depletion in response to the diacylglycerol homologue PMA in RAW 264.7 and human monocyte-derived macrophages. In these cells, we observed PPARgamma-dependent inhibition of nuclear factor-kappaB (NF-kappaB) activation and TNF-alpha expression in response to PMA. Elucidating the underlying mechanism, we found PPARgamma1 expression not only in the nucleus but also in the cytoplasm. Activation of PPARgamma1 wild type, but not an agonist-binding mutant of PPARgamma1, attenuated PMA-mediated PKCalpha cytosol to membrane translocation. Coimmunoprecipitation assays pointed to a protein-protein interaction of PKCalpha and PPARgamma1, which was further substantiated using a mammalian two-hybrid system. Applying PPARgamma1 mutation and deletion constructs, we identified the hinge helix 1 domain of PPARgamma1 that is responsible for PKCalpha binding. Therefore, we conclude that PPARgamma1-dependent inhibition of PKCalpha translocation implies a new model of macrophage desensitization.     

Permissive transmembrane helix heterodimerization is required for the expression of a functional integrin

The current paradigm is that integrin is activated via inside-out signalling when its cytoplasmic tails and TMs (transmembrane helices) are separated by specific cytosolic protein(s). Perturbations of the helical interface between the alpha- and beta-TMs of an integrin, as a result of mutations, affect its function. Previous studies have shown the requirement for specific pairing between integrin subunits by ectodomain-exchange analyses. It remains unknown whether permissive alpha/beta-TM pairing of an integrin is also required for pairing specificity and the expression of a functionally regulated receptor. We performed scanning replacement of integrin beta2-TM with a TM of other integrin beta-subunits. With the exception of beta4 substitution, others presented beta2-integrins with modified phenotypes, either in their expression or ligand-binding properties. Subsequently, we adopted alphaLbeta2 for follow-on experiments because its conformation and affinity-state transitions have been well defined as compared with other members of the beta2-integrins. Replacement of beta2- with beta3-TM generated a chimaeric alphaLbeta2 of an intermediate affinity that adhered to ICAM-1 (intercellular adhesion molecule 1) but not to ICAM-3 constitutively. Replacing alphaL-TM with alphaIIb-TM, forming a natural alphaIIb/beta3-TM pair, reversed the phenotype of the chimaera to that of wild-type alphaLbeta2. Interestingly, the replacement of alphaLbeta2- with beta3-TM showed neither an extended conformation nor the separation of its cytoplasmic tails, which are well-reported hallmarks of an activated alphaLbeta2, as determined by reporter mAb (monoclonal antibody) KIM127 reactivity and FRET (fluorescence resonance energy transfer) measurements respectively. Collectively, our results suggest that TM pairing specificity is required for the expression of a functionally regulated integrin.     

Bid is not required for Bax translocation during UV-induced apoptosis

UV irradiation triggers apoptosis through both the membrane death receptor and the intrinsic apoptotic signaling pathways. Bax, a member of the Bcl-2 family of proteins, translocates from the cytosol to the mitochondrial membrane during UV-induced apoptosis, but the regulation of Bax translocation by UV irradiation remains elusive. In this study, we show that Bax translocation, caspase-3 activation and cell death by UV irradiation are not affected by Z-IETD-fmk (caspase-8 inhibitor), but delayed by Pifithrin- (p53 inhibitor), although Bid cleavage could be completely abolished by Z-IETD-fmk. Co-transfecting YFP-Bax and Bid-CFP into human lung adenocarcinoma cells, we demonstrate that translocation of YFP-Bax precedes that of Bid-CFP, there is no significant FRET (fluorescence resonance energy transfer) between them. Similar results are obtained in COS-7 cells expressing YFP-Bax and Bid-CFP. Furthermore, using acceptor photobleaching technique, we observe that there is no interaction between YFP-Bax and Bid-CFP in both healthy and apoptotic cells. Additionally, during UV-induced apoptosis there is downregulation of Bcl-x_L, an anti-apoptotic protein. Overexpression of Bcl-x_L in cells susceptible to UV-induced apoptosis prevents Bax translocation and cell death, repression of Bid protein with siRNA (small interfering RNA) do not inhibit cell death by UV irradiation. Taken together, these data strongly suggest that Bax translocation by UV irradiation is a Bid-independent event and inhibited by overexpression of Bcl-x_L.     

Alpha-actinin-4 is selectively required for insulin-induced GLUT4 translocation

Insulin induces GLUT4 translocation to the muscle cell surface. Using differential amino acid labeling and mass spectrometry, we observed insulin-dependent co-precipitation of actinin-4 (ACTN4) with GLUT4 (Foster, L. J., Rudich, A., Talior, I., Patel, N., Huang, X., Furtado, L. M., Bilan, P. J., Mann, M., and Klip, A. (2006) J. Proteome Res. 5, 64-75). ACTN4 links F-actin to membrane proteins, and actin dynamics are essential for GLUT4 translocation. We hypothesized that ACTN4 may contribute to insulin-regulated GLUT4 traffic. In L6 muscle cells insulin, but not platelet-derived growth factor, increased co-precipitation of ACTN4 with GLUT4. Small interfering RNA-mediated ACTN4 knockdown abolished the gain in surface-exposed GLUT4 elicited by insulin but not by platelet-derived growth factor, membrane depolarization, or mitochondrial uncoupling. In contrast, knockdown of alpha-actinin-1 (ACTN1) did not prevent GLUT4 translocation by insulin. GLUT4 colocalized with ACTN4 along the insulin-induced cortical actin mesh and ACTN4 knockdown prevented GLUT4-actin colocalization without impeding actin remodeling or Akt phosphorylation, maintaining GLUT4 in a tight perinuclear location. We propose that ACTN4 contributes to GLUT4 traffic, likely by tethering GLUT4 vesicles to the cortical actin cytoskeleton.