Cell-cycle-dependent phosphorylation of the nuclear pore Nup107–160 subcomplex

Abstract

The nuclear pore complex (NPC) mediates macromolecular transport between the nucleus and the cytoplasm. Many NPC proteins (nucleoporins, Nups) are modified by phosphorylation. It is believed that phosphorylation regulates the breakdown of the nuclear envelope at mitosis and the disassembly of the NPC into different subcomplexes. In this study, we examined the cell-cycledependent phosphorylation of the Nup107-160 subcomplex, a core building block of the NPC. Using in vivo 32 P labeling in HeLa cells, we found that Nup107, Nup96, and Nup133 are phosphorylated during mitosis. To precisely map the phosphorylation sites within the complex, we used a comprehensive multiple-stage MS approach (MS, MS 2 , and MS 3 ), establishing that Nup160, Nup133, Nup96, and Nup107 are all targets of phosphorylation. We determined that the phosphorylation sites are clustered mainly at the N-terminal regions of these proteins, which are predicted to be natively disordered. In addition, we determined the cell-cycle dependence of the phosphorylation of these sites by using stable isotope labeling and MS 2 analysis. Measurement of the site-specific phosphorylation ratios between mitotic and G1 cells led us to conclude that several phosphorylation events of the subcomplex are mainly mitotic. Based on these results and our finding that the entire Nup107-160 subcomplex is stable throughout the cell cycle, we propose that phosphorylation does not affect interactions within the Nup107-160 subcomplex, but regulates the association of the subcomplex with the NPC and other proteins. mitosis ͉ nucleoporin ͉ mass spectrometry ͉ nuclear pore complex ͉ mammalian

Structural and functional analysis of Nup120 suggests ring formation of the Nup84 complex

“…These unstructured regions not only may provide flexibility for the NPC, but also may have important implications for its reversible disassembly in cells that undergo an open mitosis. In fact, the unstructured segments of the nonameric Nup107-Nup160 complex, the human homolog of the heptameric Nup84 complex, are hyperphosphorylated exclusively during mitosis (47). Here we show that an unstructured segment indeed relays the attachment between adjacent nucleoporins.…”

The discovery of a Werner helicase interacting protein (WHIP) association with the nuclear pore complex

“…These immunoprecipitated proteins were separated using a 4-20% gradient SDS-PAGE. Mass spectrometric analysis confirmed the presence of all nine members of the Nup107-160 subcomplex 24 ( Fig. 1B, Lane 1).…”

“…The proper synchronization of HeLa cells was ensured using the classic approach of double thymidine block without the addition of microtubule disruptive drugs. 24 Cells were harvested directly after release from the second thymidine block (G 1 cells) and the Nup107-160 subcomplex was immunoisolated using affinity-purified α-Nup107 antibodies. These immunoprecipitated proteins were separated using a 4-20% gradient SDS-PAGE.…”

“…The Nup107-160 subcomplex has 9 members (Nup160, Nup133, Nup107, Nup96, Nup75, Nup43, Nup37, Seh1 and Sec 13). [19][20][21][22][23][24] An additional non-Nup member may be ELYS, a putative transcription factor, which was also discovered to copurify with the Nup107-160 complex in Xenopus interphase extracts, Xenopus mitotic extracts and human cell extracts. [25][26][27] Positioned at the curvatures of the membrane embedding the NPC, the Nup107-160 subcomplex acts to stabilize these bends in the membrane.…”

The nuclear pore complex – structure and function at a glance

“…The trigger to initiate the disassembly process is the phosphorylation of several Nups by mitotic kinases (e.g. Cdk1) (Glavy et al, 2007;Laurell et al, 2011;Onischenko et al, 2005). Following their phosphorylation and release from the NPC, some nuclear pore subcomplexes also have additional roles in mitosis.…”