Bompard G et al. (2010), Subgroup II PAK-mediated phosphorylation regula...

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J Cell Biol 2010 Sep 06;1905:807-22. doi: 10.1083/jcb.200912056.

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Subgroup II PAK-mediated phosphorylation regulates Ran activity during mitosis.

Bompard G , Rabeharivelo G , Frank M , Cau J , Delsert C , Morin N .

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Ran is an essential GTPase that controls nucleocytoplasmic transport, mitosis, and nuclear envelope formation. These functions are regulated by interaction of Ran with different partners, and by formation of a Ran-GTP gradient emanating from chromatin. Here, we identify a novel level of Ran regulation. We show that Ran is a substrate for p21-activated kinase 4 (PAK4) and that its phosphorylation on serine-135 increases during mitosis. The endogenous phosphorylated Ran and active PAK4 dynamically associate with different components of the microtubule spindle during mitotic progression. A GDP-bound Ran phosphomimetic mutant cannot undergo RCC1-mediated GDP/GTP exchange and cannot induce microtubule asters in mitotic Xenopus egg extracts. Conversely, phosphorylation of GTP-bound Ran facilitates aster nucleation. Finally, phosphorylation of Ran on serine-135 impedes its binding to RCC1 and RanGAP1. Our study suggests that PAK4-mediated phosphorylation of GDP- or GTP-bound Ran regulates the assembly of Ran-dependent complexes on the mitotic spindle.

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Species referenced: Xenopus laevis
Genes referenced: birc5 cdc27 cdkn1a nsg1 pak1 pak4 plk1 ran rangap1 rcc1 stmn1 sult2a1 tpx2

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	Figure 1. Ran-GTP–induced MT aster nucleation is enhanced in X-PAK4–depleted CSF extracts. (A) Asters and mitotic spindle assembly was initiated by addition of 15 µM RanQ69L, either in ΔMBP or ΔX-PAK4 extracts. Representative fluorescent micrograph taken at low magnification showing the abundance of MTs structures formed 20 min after RanQ69L addition. Bar, 100 µm. (B) Representative micrographs of MT structures assembled as in A and after addition of recombinant X-PAK4 for complementation at indicated times. Bar, 25 µm. (C) Extracts treated as in B were analyzed by immunoblot, for X-PAK4 depletion and complementation (X-PAK4 wt) and for recombinant Ran stability (GST). β-Tubulin was used as a loading control. (D) Quantification of the numbers and quality (asters or spindles) formed in the extracts as in B (n = 3, ±SD). Control condition is given an arbitrary 100% value at each time point.
	Figure 2. X-PAK4 phosphorylates Ran on serine-135. (A and B) Autoradiographies (top panels) and Poly-His immunoblots as loading controls of the same membranes (bottom panels) of in vitro phosphorylation by active X-PAK4Ct of His-tagged wt, T24N, and Q69L Ran mutants (A); and T32A, T97A, S135A, and S153A Ran mutants (B). (C) Immunoblots of the His-tagged Ran wt and Q69L mutant after incubation with (+) or without (−) X-PAK4Ct using Ran Ser135P and Poly-His antibodies. (D) Autoradiographies (top panels), before immunoblots using Ran Ser135P and Poly-His antibodies (bottom panels), of His-tagged Ran mutants and stathmin (Sta) and of α/β casein (Cas) after phosphorylation by X-PAK4Ct (left panels) or Plx1 kinase (right panels). X-PAK4 and Plx matching panels were exposed and processed together for both autoradiography and immunoblots.
	Figure 3. Ran Ser135P concentration increases during mitosis. Ran Ser135P shares common subcellular localization with active PAK4. (A, left) Immunoblots of total HeLa cell extracts from asynchronous, G1/S, G2/M, prometaphase (0) and after nocodazole (NZ) release probed for Cdc27, survivin as mitotic markers, γ-tubulin as loading control, Ran and Ran Ser135P. Fold induction of Ran Ser135P is indicated. (A, right) FACS analyses of cells upon NZ release. (B) Localization of Ran Ser135P and active X-PAK4 Ser533P in XL-2 cells during interphase. Cells were stained for DNA immunostained with GT335 and Ran Ser135P or the active forms of subgroup II PAK (X-PAK4 Ser533P). Arrowheads show Ran Ser135P and X-PAK4 Ser533P at centrioles and lamellipodia. Bar, 25 µm.
	Figure 4. Ran Ser135P shares common subcellular localization with active PAK4. Localization of Ran Ser135P and X-PAK4 Ser533P in XL-2 cells during mitosis. Metaphase panels are shown at higher magnification. Cells were stained as in Fig. 3. β-Tubulin was immunostained instead of GT335. Bars, 10 µm (except for zoomed metaphase, 2.5 µm).
	Figure 5. The mutation of Ran serine-135 to aspartic acid (D) modulates the aster-promoting activity of GDP- but not GTP-bound RanQ69L. Asters and mitotic spindle assembly after addition of 15 µM GDP- or GTP-bound RanQ69L or RanQ69L S135A or RanQ69L S135D mutants. (A) Immunoblot analyses of total extract for β-tubulin as loading control and Ran for recombinant Ran. (B) 1 µM GDP-loaded Ran wt, S135A, and S135D mutants were mixed with 1 µM MANT-GTP. Nucleotide exchange reaction was measured in relative fluorescence units (RFU) by FRET at 460 nm. (C, top) Representative micrographs of MT structures after indicated times. Bar, 25 µm. (Bottom) Same structures assembled after 60 min of incubation were costained for TPX2. Bar, 10 µm.
	Figure 6. The mutation of Ran serine-135 to aspartic acid (D) modulates the aster-promoting activity of GDP- but not GTP-bound RanQ69L. (A) Quantification of MTs structures (asters and spindles), in RanQ69L GDP and RanQ69L GTP assays, described in Fig. 5, are respectively given an arbitrary 100% value at 20 min (n = 3, ±SD). (B) MT-associated proteins (MAPs) were purified after 40 min and TPX2 and β-tubulin levels were determined by immunoblot analyses in total extract (T), MAP-depleted extract (S), and MAP (M) fraction using specific antibodies.
	Figure 7. Active PAK4-induced phosphorylation of RanQ69L regulates the MT structures induced by the GTPase. Extracts were incubated for 15 min with GFP or GFP-tagged PAK4 E/N immobilized on protein-A Dynabeads prior aster assembly initiation by 15 µM GDP- or GTP-bound RanQ69L. (A) Representative structures observed at 20 and 40 min incubation. Bar, 25 µm. (B) Quantification of MT structures (asters or spindles) formed in the extracts as in A. GFP + GDP and GTP-bound RanQ69L conditions at 20 min are given an arbitrary 100% value. (n = 3, ±SD). (C) Extracts incubated for 40 min as in A were analyzed by immunoblotting for GFP and GFP-hPAK4 E/N (left pannel), phosphorylation of recombinant Ran proteins on serine-135 (Ran Ser135P), and of endogenous stathmin on serine 16 (Stathmin Ser16P; right panel). β-Tubulin demonstrates equal loading.
	Figure 8. RanGAP1 and RCC1 binding to Ran S135D are deficient. (A and B) Whole cell lysates prepared from HEK293 cells transfected with the indicated HA-tagged Ran mutant constructs were immunoprecipitated using HA antibody. Total lysate and immunoprecipitates were analyzed by immunoblotting for the indicated proteins. (C) Representative MT structures observed 20 min after addition of 2 µM RCC1 in presence of buffer or 15 µM Ran wt, Ran S135A, or Ran S135D. Bar, 10 µm. (D) 1 µM GDP-loaded recombinant Ran wt, S135A, and S135D mutants were added to a mixture of recombinant RCC1 and 1 µM MANT-GTP. A representative experiment of the exchange reaction that was measured as described in Fig. 5 B (n = 5).
	Figure 9. PAK4 depletion induces a G2/M block. (A) Representative immunoblot analyses of protein extracts from asynchronous (NZ−) or prometaphase-enriched (NZ+) HeLa cells transfected with luciferase or PAK4 siRNAs using indicated antibodies. (B) Quantification of Ran Ser135P present in the cells as in A (n = 7, ±SEM). (C) Mitotic index of PAK4- and luciferase-depleted cells after release from a G2/M block induced by RO3306 (n = 3, ±SEM) and four microscopic fields of β-tubulin staining of a representative experiment after 30 min release. Bar, 25 µm.
	Figure 10. PAK4 depletion induces a G2/M block. (A) Percentage of 2N cells determined by FACS analysis fom a representative experiment (n = 4) described in Fig. 9 C and four microscopic fields of β-tubulin staining after 120 min release from the G2/M block. Bar, 50 µm. Number of 2N cells at 300 min in luciferase-depleted cells was given an arbitrary 100% value. A representative experiment, out of four independent experiments, is shown. (B) Total cell lysates from cells treated as described above were analyzed by immunoblot using indicated antibodies.

References [+] :

Arias-Romero, A tale of two Paks. 2008, Pubmed