Phosphorylation of RanGAP1 Stabilizes Its Interaction with Ran and RanBP1

Abstract

ABSTRACT. Ran is a nuclear Ras-like GTPase that is required for various nuclear events including the bi-directional transport of proteins and ribonucleoproteins through the nuclear pore complex, spindle formation, and reassembly of the nuclear envelope. One of the key regulators of Ran is RanGAP1, a Ran specific GTPase activating protein. The question of whether a mechanism exists for controlling nucleocytoplasmic transport through the regulation of RanGAP1 activity continues to be debated. Here we show that RanGAP1 is phosphorylated in vivo and in vitro. Serine-358 ( 358 S) was identified as the major phosphorylation site, by MALDI-TOF-MS spectrometry. Site directed mutagenesis at this position abolished the phosphorylation. Experiments using purified recombinant kinase and specific inhibitors such as DRB and apigenin strongly suggest that casein kinase II (CK2) is the responsible kinase. Although the phosphorylation of 358 S of RanGAP1 did not significantly alter its GAP activity, the phosphorylated wild type RanGAP1, but not a mutant harboring a mutation at the phosphorylation site 358 S, efficiently formed a stable ternary complex with Ran and RanBP1 in vivo, suggesting that the 358 S phosphorylation of RanGAP1 affects the Ran system.

An ARF6–Exportin-5 axis delivers pre-miRNA cargo to tumour microvesicles

“…Following nuclear export, GTP hydrolysis on Ran is facilitated by the GTPase-activating protein, RanGAP1, which liberates cargo for further transport 48 . Given the previously reported activation of casein kinase 2 (CK2) downstream of ARF6-GTP 49 and literature documenting that CK2-mediated 358 S phosphorylation of RanGAP1 allows RanGAP1 to form a stable ternary complex with Ran-GTP and RanBP1 50 , we examined the role of CK2 in Exportin-5 and pre-miRNA trafficking to shed TMVs. Treatment with the small molecule inhibitor of CK2 4,5,6,7-tetrabromobenzotriazole (TBB), reduces Exportin-5 included as shed TMV cargo (Figure 4a), along with a concomitant reduction in TMV pre-miRNA, and miRNA cargo (Figure 4b, Supplementary Figure 3i).…”

The Mobile FG Nucleoporin Nup98 Is a Cofactor for Crm1-dependent Protein Export

“…GST-Nup98, GST-Nup214 (aa 1864-2090), GST-Crm1, and GST-RanBP3 fusion constructs were generated by insertion of the appropriate PCR fragments into the pGEX6P vector (GE Healthcare, Piscataway, NJ). GST-Nup98 fusion protein–encoding plasmids were prepared by subcloning the Nup98 fragments in-frame with GST in pCAG-wtag, which contains GST under the control of the CAG promoter (Takeda et al , 2005). The Nup98FG-EGFP encoding plasmid (pNup98FG-EGFP) was constructed by inserting the appropriate PCR fragment of Nup98 (aa 1-480) into the pEGFP-N1 vector (Clontech).…”

Iron and FER‐LIKE IRON DEFICIENCY‐INDUCED TRANSCRIPTION FACTOR‐dependent regulation of proteins and genes in Arabidopsis thaliana roots

“…Among the multiply found proteins, RANGAP1 (AT3G63130) plays a role in the translocation of proteins through the nuclear pore complex and is associated with cell plate and mitotic spindle formation . In humans, the NPC‐associated form of RANGAP1 is SUMOylated and phosphorylated . In case the same mechanisms apply to Arabidopsis, the two RANGAP1 spots might represent differently phosphorylated forms, which would be supported by the observed difference in p I rather than MW.…”