Signals and Pathways Regulating Nucleolar Retention of Novel Putative Nucleolar GTPase NGP-1(GNL-2)

Abstract

NGP-1(GNL-2) is a putative GTPase, overexpressed in breast carcinoma and localized in the nucleolus. NGP-1 belongs to the MMR1-HSR1 family of large GTPases that are emerging as crucial coordinators of signaling cascades in different cellular compartments. The members of this family share very closely related G-domains, but the signals and pathways regulating their subcellular localization and their functional relevance remain unknown. To improve our understanding of the nuclear transport mechanism of NGP-1, we have identified two nucleolar localization signals (NoLS) that are independently shown to translocate NGP-1 as well the heterologous protein to the nucleolus. Site-specific mutagenesis and immunofluorescence studies suggest that the tandem repeats of positively charged amino acids are critical for NGP-1 NoLS function. Interestingly, amino-terminal (NGP-1(1-100)) and carboxyl-terminal (NGP-1(661-731)) signals independently interact with receptors importin-β and importin-α, respectively. This investigation, for the first time, provides evidence that the interaction of importin-α with C-terminal NoLS (NGP-1(661-731)) was able to target the heterologous protein to the nucleolar compartment. Structural modeling analysis and alanine scanning mutagenesis of conserved G-domains suggest that G4 and G5 motifs are critical for GTP binding of NGP-1 and further show that the nucleolar localization of NGP-1 is regulated by a GTP gating-mediated mechanism. In addition, our data suggest that an ongoing transcription is essential for efficient localization of NGP-1 to the nucleolus. We have observed a high level of NGP-1 expression in the mitogen-activated primary human peripheral blood mononuclear cells (hPBMC) as well as in human fetal brain-derived neural precursor cells (hNPCs) in comparison to cells undergoing differentiation. Overall, the results suggest that multiple mechanisms are involved in the localization of NGP-1 to the nucleolus for the regulation of nucleolar function in cell growth and proliferation.

Nucleolar GTP-binding Protein-1 (NGP-1) Promotes G1 to S Phase Transition by Activating Cyclin-dependent Kinase Inhibitor p21Cip1/Waf1

“…Studies using zebrafish showed p53 stabilization upon Ngp-1 depletion (17) and also suggested its role in ribosome biogenesis (18). Previous studies from our laboratory showed that increased NGP-1 expression in human neural precursor cells compared with differentiated astrocytes (16). Furthermore, NGP-1 levels were up-regulated in mitogeninduced human peripheral blood mononuclear cells (16), suggesting its association with cell proliferation.…”

“…Previous studies from our laboratory showed that increased NGP-1 expression in human neural precursor cells compared with differentiated astrocytes (16). Furthermore, NGP-1 levels were up-regulated in mitogeninduced human peripheral blood mononuclear cells (16), suggesting its association with cell proliferation. Nevertheless, the mechanism by which NGP-1 modulates cell proliferation remains unexplored.…”

“…Plasmid Construction-NGP-1-GFP and its deletion constructs (NGP-1(1-100), NGP-1(101-600), and NGP-1(601-731)) were generated as described elsewhere (16). Lyar and RPL23A were amplified from the HEK-293T cDNA library using appropriate primers (Table 1) and cloned as GST fusion in the pGEX-4T-1 vector.…”

“…Protein-Protein Interaction Assays-For GST pulldown assay, cell lysates with equal amounts of protein were incubated with glutathione-Sepharose beads pre-bound with GST-RPL23A or GST-LYAR as described elsewhere (16). For coimmunoprecipitation, equal amounts of cell lysates were incubated with appropriate antibody, and the bound protein complexes were eluted and resolved on SDS-PAGE followed by Western blotting with appropriate antibodies.…”

“…Another member of the same family, guanine nucleotide-binding protein-like 2 (GNL2) also known as nucleolar GTP-binding protein-1 (NGP-1), is observed to be overexpressed in breast, colon, and colorectal cancers and is a breast cancer autoantigen (15). It shuttles between the nucleolus and nucleoplasm (11), and its nucleolar retention is a multifactordependent process (16). Studies using zebrafish showed p53 stabilization upon Ngp-1 depletion (17) and also suggested its role in ribosome biogenesis (18).…”