Importin α/β and Ran-GTP Regulate XCTK2 Microtubule Binding through a Bipartite Nuclear Localization Signal

Abstract

The small GTPase Ran is essential for spindle assembly. Ran is proposed to act through its nuclear import receptors importin ␣ and/or importin ␤ to control the sequestration of proteins necessary for spindle assembly. To date, the molecular mechanisms by which the Ran pathway functions remain unclear. Using purified proteins, we have reconstituted Ran-regulated microtubule binding of the C-terminal kinesin XCTK2, a kinesin important for spindle assembly. We show that the tail of XCTK2 binds to microtubules and that this binding is inhibited in the presence of importin ␣ and ␤ (␣/␤) and restored by addition of Ran-GTP. The bipartite nuclear localization signal (NLS) in the tail of XCTK2 is essential to this process, because mutation of the NLS abolishes importin ␣/␤-mediated regulation of XCTK2 microtubule binding. Our data show that importin ␣/␤ directly regulates the activity of XCTK2 and that one of the molecular mechanisms of Ran-regulated spindle assembly is identical to that used in classical NLS-driven nuclear transport. INTRODUCTIONThe process of chromosome congression and segregation is mediated by the mitotic spindle, which is comprised of microtubules (MTs) and their associated proteins. Proper spindle assembly requires the activities of both plus-endand minus-end-directed MT motors, nonmotor MT-associated proteins, and other essential non-MT-associated proteins (Compton, 2000). Minus-end-directed cytoplasmic dynein and plus-end-directed Eg5 play key roles in the organization of spindle MTs in Xenopus egg extracts and in cells (Gaglio et al., 1996;Heald et al., 1996;Merdes et al., 1996;Walczak et al., 1998). In addition, the mitotic minus-enddirected C-terminal kinesins function as MT cross-linkers to promote proper spindle assembly in many organisms (Endow and Komma, 1996;Walczak et al., 1997Walczak et al., , 1998Matuliene et al., 1999;Mountain et al., 1999;Ovechkina and Wordeman, 2003) and KIFC1 and KIFC5 C-terminal kinesins may serve a similar cross-linking function during spermatogenesis (Navolanic and Sperry, 2000;Yang and Sperry, 2003). The mechanism that regulates this MT cross-linking is currently unknown.Recently, the GTPase Ran was shown to be sufficient to induce MT aster formation as well as bipolar spindle assembly in egg extracts (Carazo-Salas et al., 1999;Kalab et al., 1999;Ohba et al., 1999;Wilde and Zheng, 1999;Nachury et al., 2001). Subsequently, it was reported to be involved in regulating numerous mitotic spindle assembly processes, including MT nucleation and dynamics (Wilde and Zheng, 1999;Carazo-Salas et al., 2001;Wilde et al., 2001), MT motor activity , and nuclear envelope assembly (Bamba et al., 2002;Hetzer et al., 2002;Zhang et al., 2002). It is believed that Ran provides positional cues by the generation of a steep gradient of Ran-GTP between the condensed chromosomes and the surrounding cytoplasm (Kalab et al., 2002;Trieselmann and Wilde, 2002). After nuclear envelope breakdown, the localized concentration of Ran-GTP around chromatin is thought to contribute to proper spind...

Kinesin-14 Family Proteins HSET/XCTK2 Control Spindle Length by Cross-Linking and Sliding Microtubules

“…pEGFP-HSET and pEGFP-HSET N593K clones were generated by subcloning their cDNA into the pEGFPC1 vector by EcoRI/SalI. The XCTK2 constructs and mutants were described previously (Ems-McClung et al, 2004). All resulting clones were verified by sequencing.…”

“…GST-HSET(2-304) was expressed in Escherichia coli BL21(DE3) cells and affinity-purified on glutathione Sepharose 4B (Amersham Biosciences, Piscataway, NJ) as previously described (Ems-McClung et al, 2004), followed by the gel filtration on a Superose 12 column (GE Healthcare Life Sciences, Piscataway, NJ) equilibrated in column buffer (50 mM MOPS, pH 7.2, 50 mM NaCl, 0.1 mM EDTA, and 0.1 mM EGTA). Fractions were eluted, and sucrose was added to 10%.…”

“…Immunoprecipitations from Xenopus egg extracts were performed as described previously (Ems-McClung et al, 2004). Anti-GFP and nonimmune rabbit IgG were covalently coupled to the Affi-prep protein A beads (Bio-Rad; Harlow and Lane, 1999).…”

“…One important Ran-regulated factor needed for proper spindle assembly is the Kinesin-14 family member XCTK2 (Walczak et al, 1997;Ems-McClung et al, 2004). Kinesin-14 family members are minus-end-directed MT motors (McDonald et al, 1990;Walker et al, 1990) that are needed for spindle assembly and pole organization in both mitotic and meiotic systems (Hatsumi and Endow, 1992a,b;Endow and Komma, 1996;Walczak et al, 1997;Matuliene et al, 1999;Mountain et al, 1999;Sharp et al, 1999;Goshima et al, 2005a).…”

“…The motor domain binds MTs in an ATP-sensitive manner, and the tail possesses a second ATP-insensitive MT binding site that enables Kinesin-14s to cross-link and slide MTs (Chandra et al, 1993;Walker, 1995;Walczak et al, 1997;Karabay and Walker, 1999;Ems-McClung et al, 2004). We showed previously that XCTK2 is regulated by Ran via the association of a bipartite NLS in the tail of XCTK2 with importin ␣/␤ (Ems- McClung et al, 2004). Binding of importin ␣/␤ to XCTK2 inhibits its association with MTs and blocks its ability to cross-link MTs.…”