Estradiol dependent anchoring of the goat uterine estrogen receptor activation factor (E-RAF) at the endoplasmic reticulum by a 55 kDa anchor protein (ap55)

The primary intracellular site of localization of the estrogen receptor activation factor (E-RAF) is shown here to be the endoplasmic reticulum where the protein remains anchored through an estrogen dependent mechanism. The retention of E-RAF by the endoplasmic reticulum is facilitated by two proteins: (1) a 55 kDa anchor protein (ap55) which is an integral membrane protein of the endoplasmic reticulum. ap55 is a high affinity estrogen binding protein. A conformational change induced by estrogen binding is thought to favor the anchoring process. (2) The anchoring of E-RAF by ap55 is mediated by yet another protein. This is the 66 kDa transport protein (tp66) which recognizes ap55 on the one hand and E-RAF on the other. The presence of estradiol that saturates the hormone binding sites on ap55 appears to favor the anchoring of tp66-E-RAF complex to ap55. This interaction appears to be weakened by levels of estradiol below 7 nM concentration leading to the dissociation of the tp66-E-RAF complex from ap55. The tp66-E-RAF complex moves towards the nucleus.     

Proteins which mediate the nuclear entry of goat uterine non activated estrogen receptor (naER) following naER internalization from the plasma membrane

The nuclear transport of the internalised naER is influenced by a 58 kDa protein, p58, that appears to recognize the nuclear localization signals on the naER. At the nuclear pore complex the naER-p58 complex binds to a 62 kDa protein, p62; p58 recognizes p62 in this interaction. It is further observed that p62 gets 'docked' at a 66 kDa nuclear pore complex protein, npcp66. The nuclear entry of naER is an ATP-dependent process. An ATP-dependent biphasic nuclear entry of naER, has been observed. It is possible that the docking of p58-naER complex at the nuclear pore complex and the eventual nuclear entry of naER following its dissociation from the p58 are influenced by two different ranges in the concentration of ATP. In this process, it appears that, the nuclear entry requires an additional quantum of energy, provided by the hydrolysed ATP, in contrast to the energy requirement associated with, the nuclear 'docking' event.     

The p55 subunit of Drosophila chromatin assembly factor 1 is homologous to a histone deacetylase-associated protein.

To gain a better understanding of DNA replication-coupled chromatin assembly, we have isolated the cDNA encoding the smallest (apparent molecular mass, 55 kDa; termed p55) subunit of Drosophila melanogaster chromatin assembly factor 1 (dCAF-1), a multisubunit protein that is required for the assembly of nucleosomes onto newly replicated DNA in vitro. The p55 polypeptide comprises seven WD repeat motifs and is homologous to the mammalian RbAp48 protein, which is associated with the HD1 histone deacetylase. dCAF-1 was immunopurified by using affinity-purified antibodies against p55; the resulting dCAF-1 preparation possessed the four putative subunits of dCAF-1 (p180, p105, p75, and p55) and was active for DNA replication-coupled chromatin assembly. Moreover, dCAF-1 activity was specifically depleted with antibodies against p55. Thus, p55 is an integral component of dCAF-1. p55 is localized to the nucleus and is present throughout Drosophila development. Consistent with the homology between p55 and the HD1-associated RbAp48 protein, histone deacetylase activity was observed to coimmunoprecipitate specifically with p55 from a Drosophila nuclear extract. Furthermore, a fraction of the p55 protein becomes associated with the newly assembled chromatin following DNA replication. These findings collectively suggest that p55 may function as a link between DNA replication-coupled chromatin assembly and histone modification.     

Characterization of the 90 kDa heat shock protein (HSP90)-associated ATP/GTPase

The 90 kDa heat shock protein (HSP90) is an ATP-binding molecular chaperone with an associated ATPase activity having nucleoplasmin and HSP70-binding homology domains and containing Ca-binding EF-hands and a nuclear localization signal. Here we characterize the HSP90-associated ATPase and show that it is (i) a P-type ATPase inhibited by molybdate and vanadate, (ii) able to hydrolyze methylfluorescein phosphate with a 5–6-fold higher affinity, (iii) a 3-times better GTPase than ATPase in the presence of calcium and (iv) HSP27 and F-actin, but not HSP10 can “convert” the HSP90-associated ATPase activity to HSP90 autokinase activity. The HSP90-associated ATP/GTPase may participate in the regulation of complex formation of HSP90 with other proteins, such as F-actin, tubulin and heat shock proteins.     

Internuclear exchange of an inner nuclear membrane protein (p55) in heterokaryons: in vivo evidence for the interaction of p55 with the nuclear lamina

The movement between nuclei of an integral protein of the inner nuclear membrane has been studied in rat/mouse and rat/hamster heterokaryons. This protein, p55, was found to equilibrate between nuclei over a period of approximately 6 h in the absence of new protein synthesis. When rat/mouse heterokaryons were constructed using an undifferentiated murine embryonal carcinoma (P19), which lacks lamins A and C, no accumulation of p55 in the mouse cell nucleus was observed. However, P19 nuclei could be rendered competent to accumulate p55 by transfecting the parent cells with human lamin A before cell fusion, supporting the notion that p55 may interact with the nuclear lamina. Since p55 does not appear to be able to dissociate from the nuclear membrane, it is concluded that this exchange between nuclei does not occur in the aqueous phase and instead is probably membrane mediated. It is proposed that this protein may be free to move between the inner and outer nuclear membranes via the continuities at the nuclear pore complexes and that transfer between nuclei occurs via lateral diffusion through the peripheral ER, which appears to form a single continuous membrane system in these heterokaryons. One implication of these observations is that accumulation of at least some integral proteins in the inner nuclear membrane may be mediated by interactions with other nuclear components and may not require a single defined targeting sequence.     

The DEAD-box protein Dbp5p is required to dissociate Mex67p from exported mRNPs at the nuclear rim

Eukaryotic mRNAs are exported from the nucleus to the cytoplasm as complex mRNA-protein particles (mRNPs), and translocation through the nuclear pore complex (NPC) is accompanied by extensive structural changes of the mRNP. We have tested the hypothesis that the DEAD-box ATPase Dbp5p is required for such an mRNP rearrangement. In dbp5 mutant cells, the mRNA export receptor Mex67p accumulates on mRNA. This aberrant accumulation of Mex67p with RNA and the cold-sensitive growth phenotype of a dbp5 allele are suppressed by a mex67 mutation. Moreover, Mex67 bound mRNA accumulates at the nuclear rim in a temperature-sensitive dbp5 mutant when the nuclear exosome is impaired. Importantly, although accumulation of Mex67p-containing mRNPs is also observed when a nuclear basket component is mutated, these mRNPs still contain the nuclear export factor Yra1p. In contrast, the dbp5-trapped mRNPs lack Yra1p. We propose that Dbp5p's function is specifically required to displace Mex67p from exported mRNPs, thus terminating export.     

A novel nuclear pore protein Nup82p which specifically binds to a fraction of Nsp1p

Nsp1p interacts with nuclear pore proteins Nup49p, Nup57p and Nic96p in a stable complex which participates in nucleocytoplasmic transport. An additional p80 component is associated with Nsp1p, but does not co- purify with tagged Nup57p, Nup49p and Nic96p. The p80 gene was cloned and encodes a novel essential nuclear pore protein named Nup82p. Immunoprecipitation of tagged Nup82p reveals that it is physically associated with a fraction of Nsp1p which is distinct from Nsp1p found in a complex with Nup57p, Nic96p and Nup49p. The Nup82 protein can be divided into at least two different domains both required for the essential function, but it is only the carboxy-terminal domain, exhibiting heptad repeats, which binds to Nsp1p. Yeast cells depleted of Nup82p stop cell growth and concomitantly show a defect in poly(A)+RNA export, but no major alterations of nuclear envelope structure and nuclear pore density are seen by EM. This shows that Nsp1p participates in multiple interactions at the NPC and thus has the capability to physically interact with different NPC structures.     

The yeast nucleoporin Nup53p specifically interacts with Nic96p and is directly involved in nuclear protein import

The bidirectional nucleocytoplasmic transport of macromolecules is mediated by the nuclear pore complex (NPC) which, in yeast, is composed of approximately 30 different proteins (nucleoporins). Pre-embedding immunogold-electron microscopy revealed that Nic96p, an essential yeast nucleoporin, is located about the cytoplasmic and the nuclear periphery of the central channel, and near or at the distal ring of the yeast NPC. Genetic approaches further implicated Nic96p in nuclear protein import. To more specifically explore the potential role of Nic96p in nuclear protein import, we performed a two-hybrid screen with NIC96 as the bait against a yeast genomic library to identify transport factors and/or nucleoporins involved in nuclear protein import interacting with Nic96p. By doing so, we identified the yeast nucleoporin Nup53p, which also exhibits multiple locations within the yeast NPC and colocalizes with Nic96p in all its locations. Whereas Nup53p is directly involved in NLS-mediated protein import by its interaction with the yeast nuclear import receptor Kap95p, it appears not to participate in NES-dependent nuclear export.     

Siglec-15 protein regulates formation of functional osteoclasts in concert with DNAX-activating protein of 12 kDa (DAP12)

Osteoclasts are multinucleated giant cells that reside in osseous tissues and resorb bone. Signaling mediated by receptor activator of nuclear factor (NF)-κB (RANK) and its ligand leads to the nuclear factor of activated T cells 2/c1 (NFAT2 or NFATc1) expression, a critical step in the formation of functional osteoclasts. In addition, adaptor proteins harboring immunoreceptor tyrosine-based activation motifs, such as DNAX-activating protein of 12 kDa (DAP12), play essential roles. In this study, we identified the gene encoding the lectin Siglec-15 as NFAT2-inducible, and we found that the protein product links RANK ligand-RANK-NFAT2 and DAP12 signaling in mouse osteoclasts. Both the recognition of sialylated glycans by the Siglec-15 V-set domain and the association with DAP12 through its Lys-272 are essential for its function. When Siglec-15 expression was knocked down, fewer multinucleated cells developed, and those that did were morphologically contracted with disordered actin-ring structures. These changes were accompanied by significantly reduced bone resorption. Siglec-15 formed complexes with Syk through DAP12 in response to vitronectin. Furthermore, chimeric molecules consisting of the extracellular and transmembrane regions of Siglec-15 with a K272A mutation and the cytoplasmic region of DAP12 significantly restored bone resorption in cells with knocked down Siglec-15 expression. Together, these results suggested that the Siglec-15-DAP12-Syk-signaling cascade plays a critical role in functional osteoclast formation.     

Phosphorylation of the membrane proximal region of tumor necrosis factor receptor CD120a (p55) at ERK consensus sites

The interaction of tumor necrosis factor-alpha with its receptor CD120a (p55) initiates downstream signaling cascades that include the activation of the mitogen-activated protein kinase (MAPK), p42(mapk/erk2). The membrane proximal region of CD120a (p55) is Ser-, Thr-, and Pro-rich and contains four mitogen-activated protein kinase consensus phosphorylation sites. In recent work, we showed that CD120a (p55) itself is a target of phosphorylation by p42(mapk/erk2), and after phosphorylation, the receptor is redistributed from the cell surface and Golgi complex to intracellular tubular structures associated with elements of the endoplasmic reticulum. The goal of this study was to define the specific amino acid residues that are phosphorylated. Deletional mutagenesis of the cytoplasmic domain of CD120a (p55) indicated that two sites located between residues 207-254 and 250-300 were phosphorylated predominantly on Thr and Ser residues, respectively. Site-directed mutagenesis of Ser and Thr residues contained within the extracellular signal-regulated kinase (ERK) consensus sequences indicated that the preferred residues were Thr-236 and Ser-270. Primary phosphorylation at these sites appeared to enable subsequent phosphorylation at Ser-240 and Ser-244, although the level of phosphorylation of these latter two sites was less than the preferred sites. Through the use of specific ligation of CD120a (p55) alone and mice deficient in CD120a (p55), CD120b (p75), or both receptors, CD120a (p55) was shown to be necessary and sufficient for the induction of kinase activity. These findings thus suggest that the phosphorylation of Thr-236 and Ser-270 within the membrane proximal region of CD120a (p55) are the preferred sites of phosphorylation by p42(mapk/erk2) and may set in motion phosphorylation at other sites.     

Nuclear estrogen receptor II (nER-II) is involved in the estrogen-dependent ribonucleoprotein transport in the goat uterus: II. Isolation and characterization of three small nuclear ribonucleoprotein proteins which bind to nER-II.

Three proteins of a goat uterine small nuclear ribonucleoprotein (snRNP) fraction, which bind to nuclear estrogen receptor-II (nER-II) have been isolated and purified. These are the p32, p55, and p60 of which p32 is the major nER-II binding protein. Indirect evidence reveals that p32 binds to the nuclear export signal (NES) on the nER-II. nER-II is a snRNA binding protein while p32 does not bind to the RNA. nER-II along with p32 and p55 form an effective Mg(++)ATPase complex, the activation of which appears to be the immediate reason behind the RNP exit from the nuclei following estradiol exposure. The three nER-II binding proteins bind to the nuclear pore complex; nER-II does not possess this property.     

p75(NGFR) and TrkA receptors collaborate to rapidly activate a p75(NGFR)-associated protein kinase.

The role of the low affinity nerve growth factor receptor (p75(NGFR)) in NGF-mediated signaling is not yet understood. Here we show by co-immunoprecipitation that NGF activates a protein kinase that is directly associated with p75(NGFR) in dorsal root ganglion (DRG) cells and PC12 cells in culture. Two proteins of 120 and 104 kDa constitute the majority of this activity. In PC12 cells, TrkA activation was necessary to elicit p75(NGFR)-associated kinase activity. Although NGF binding to p75(NGFR) was not necessary for kinase activation, it accelerated the activation of the kinase at low NGF concentrations. Deletion analysis showed that a 43 amino acid region in the cytoplasmic domain of p75(NGFR) was responsible for this effect. These findings show that p75(NGFR) accelerates TrkA-mediated signaling and, in addition, demonstrate that p75NGFR and TrkA collaborate to activate a previously undescribed p75(NGFR)-associated protein kinase.     

Increase in a 55-kDa keratin-like protein in the nuclear matrix of rat liver cells during proliferative activation

We have identified a protein (p55) with a molecular weight of 55 kDa and a pI of 6.2, which was strongly increased in the nuclear matrix of rat liver cells during proliferative activation. This protein is highly insoluble since it could not be solubilized either by detergents or by alkaline extraction. We have obtained three partial amino acid sequences which revealed that p55 has a high homology with cytokeratins. Polyclonal antibodies raised against p55 were used to carry out Western blot and immunocytochemical studies which indicated that p55 was localized only in the nuclei, specifically in the nuclear matrix. Autoradiographic experiments revealed that not all the cells presenting an increase in p55 incorporated [3H]thymidine, indicating that this protein is not related to DNA replication. Immunocytochemical studies also revealed that during mitosis p55 is localized surrounding the chromosomes and associated with the mitotic apparatus, suggesting that p55 is involved in the separation of chromosomes during cell division.     

The yeast integral membrane protein Apq12 potentially links membrane dynamics to assembly of nuclear pore complexes

Although the structure and function of components of the nuclear pore complex (NPC) have been the focus of many studies, relatively little is known about NPC biogenesis. In this study, we report that Apq12 is required for efficient NPC biogenesis in Saccharomyces cerevisiae. Apq12 is an integral membrane protein of the nuclear envelope (NE) and endoplasmic reticulum. Cells lacking Apq12 are cold sensitive for growth, and a subset of their nucleoporins (Nups), those that are primarily components of the cytoplasmic fibrils of the NPC, mislocalize to the cytoplasm. APQ12 deletion also causes defects in NE morphology. In the absence of Apq12, most NPCs appear to be associated with the inner but not the outer nuclear membrane. Low levels of benzyl alcohol, which increases membrane fluidity, prevented Nup mislocalization and restored the proper localization of Nups that had accumulated in cytoplasmic foci upon a shift to lower temperature. Thus, Apq12p connects nuclear pore biogenesis to the dynamics of the NE.