Brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1) is an Ϸ200-kDa brefeldin A-inhibited guanine nucleotideexchange protein that preferentially activates ADP-ribosylation factor 1 (ARF1) and ARF3. BIG1 was found in cytosol in a multiprotein complex with a similar ARF-activating protein, BIG2, which is also an A kinase-anchoring protein. In HepG2 cells growing with serum, BIG1 was primarily cytosolic and Golgi-associated. After incubation overnight without serum, a large fraction of endogenous BIG1 was in the nuclei. By confocal immunofluorescence microscopy, BIG1 was localized with nucleoporin p62 at the nuclear envelope (probably during nucleocytoplasmic transport) and also in nucleoli, clearly visible against the less concentrated overall matrix staining. BIG1 was also identified by Western blot analyses in purified subnuclear fractions (e.g., nucleoli and nuclear matrix). Antibodies against BIG1, nucleoporin, or nucleolin coimmunoprecipitated the other two proteins from purified nuclei. In contrast, BIG2 was not associated with nuclear BIG1. Also of note, ARF was never detected among proteins precipitated from purified nuclei by anti-BIG1 antibodies, although microscopically the two proteins do appear sometimes to be colocalized in the nucleus. These data are consistent with independent intracellular movements and actions of BIG1 and BIG2, and they are also evidence of the participation of BIG1 in both Golgi and nuclear functions.
Antibodies against BIG1 or nucleolin coprecipitated both proteins from nuclei, which was abolished by the incubation of nuclei with RNase A or DNase, indicating that the interaction depended on nucleic acids. 32 P labeling of RNAs immunoprecipitated with BIG1 or nucleolin from nuclei revealed bands of Ϸ210 bases that also hybridized with U3 small nucleolar (sno)RNA-specific oligonucleotides. Clones of U3 snoRNA cDNAs from the material precipitated by antibodies against BIG1 or nucleolin yielded identical nucleotide sequences that also were found in genomic DNA. Later analyses revealed the presence of fibrillarin, nucleoporin p62, and La in BIG1 and nucleolin immunoprecipitates. Our data demonstrate that BIG1, nucleolin, U3, the U3-binding protein fibrillarin, and the RNA-binding protein La may exist together in nuclear complexes, consistent with a potential role for BIG1 in nucleolar processes. Evidence that BIG1 and nucleolin, but not fibrillarin, can be present with p62 at the nuclear envelope confirms the presence of BIG1 and nucleolin in dynamic molecular complexes that change in composition while moving through nuclei. Nuclear functions of BIG1 remain to be determined.ADP-ribosylation factor ͉ nucleolus ͉ small RNA B IG1, a brefeldin A (BFA)-inhibited guanine nucleotideexchange protein (GEP), activates class I ADP-ribosylation factors (ARF1-3) by catalyzing the replacement of ARF-bound GDP by GTP (1-3). This action is critical for the regulation of protein transport in eukaryotic cells (reviewed in ref. 4). In addition to its ARF-activating capacity, BIG1 has scaffolding functions and interactions with several proteins in other cellular compartments. We had reported interaction of BIG1 with FK506-binding protein 13 (FKBP13) and nuclear accumulation of BIG1 in cells incubated briefly with the immunosuppressive drug, FK506 (5, 6). BIG1 also contains an A-kinase-anchoring protein (AKAP) sequence identical to one of three such sequences with different specificities for binding each of the four kinase regulatory (R) subunits that were identified in BIG2 (7), a structurally similar GEP originally copurified with BIG1. PKA-catalyzed phosphorylation of a predicted substrate serine in BIG1 was later shown to result in its nuclear accumulation, which also required the nuclear localization signal that is situated in the Sec 7 domain region in BIG1 that is responsible for ARF activation (8). Subsequent in vitro experiments with recombinant proteins demonstrated that GEP activity of BIG1 was decreased after phosphorylation by PKA (9). Whether or how the cAMPinduced accumulation of BIG1 in nuclei resembles that seen earlier in cells exposed briefly to FK506 or to serum deprivation for 16-18 h (6) remains to be determined.BIG1 appeared concentrated in nucleoli of serum-deprived cells, and antibodies against BIG1 precipitated both nucleolin and BIG1 from nuclear lysates (6). Treatment of nuclei with RNase or DNase prevented coimmunoprecipitation (co-IP) of the two proteins, which led us to investigate potential nucleic aci...
Lymphangiogenesis and angiogenesis are processes that are, in part, regulated by vascular endothelial growth factor (VEGF)-D. The formation of lymphatic structures has been implicated in multiple lung diseases, including pulmonary fibrosis. VEGF-D is a secreted protein produced by fibroblasts and macrophages, which induces lymphangiogenesis by signaling via VEGF receptor-3, and angiogenesis through VEGF receptor-2. VEGF-D contains a central VEGF homology domain, which is the biologically active domain, with flanking N- and C-terminal propeptides. Full-length VEGF-D (∼ 50 kD) is proteolytically processed in the extracellular space, to generate VEGF homology domain that contains the VEGF-D receptor-binding sites. Here, we report that, independent of its cell surface receptors, full-length VEGF-D accumulated in nuclei of fibroblasts, and that this process appears to increase with cell density. In nuclei, full-length VEGF-D associated with RNA polymerase II and c-Myc. In cells depleted of VEGF-D, the transcriptionally regulated genes appear to be modulated by c-Myc. These findings have potential clinical implications, as VEGF-D was found in fibroblast nuclei in idiopathic pulmonary fibrosis, a disease characterized by fibroblast proliferation. These findings are consistent with actions of full-length VEGF-D in cellular homeostasis in health and disease, independent of its receptors.
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