Association of guanine nucleotide-exchange protein BIG1 in HepG2 cell nuclei with nucleolin, U3 snoRNA, and fibrillarin

Abstract

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...

ARF1 controls proliferation of breast cancer cells by regulating the retinoblastoma protein

“…ARF guanine-nucleotide exchange factors such as BRAG2 have been detected in nuclei both biochemically and morphologically (Dunphy et al, 2007). Moreover, both BIG1, a guanine-nucleotide exchange factor that is localized to the trans-Golgi network at steady state, and Centaurin a1, a GTPase-activating protein for ARF6, have been reported to enter the nucleus and interact with the nucleolar protein nucleolin (Dubois et al, 2003;Padilla et al, 2008). Similarly, PIKE (AGAP2/Centaurin-g1) was also found within nuclei (Dunphy et al, 2007).…”

Interaction of brefeldin A-inhibited guanine nucleotide-exchange protein (BIG) 1 and kinesin motor protein KIF21A

“…Later experiments with recombinant proteins in vitro demonstrated that GEP activity of BIG1 was decreased after phosphorylation by PKA (12). Most recently, it was reported that BIG1, nucleolin, U3 small nucleolar RNA, 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 function (13). The C-terminal region of BIG1 includes a binding site for myosin IXb.…”

Structurally Constrained Residues Outside the Binding Motif Are Essential in the Interaction of 14-3-3 and Phosphorylated Partner

“…Cell immunofluorescence was imaged using a confocal fluorescence microscope (LSM 510, Zeiss) as previously described. 18 necessary but not enough for the binding. These new insights into the conformational propensities of 14-3-3 proteins and intrinsically disordered partners reveal that other structural clues (e.g., anchor-type residues) need to be identified to understand their interactions.…”