Cdk11 is a RanGTP-dependent microtubule stabilization factor that regulates spindle assembly rate

Abstract

Production of Ran–guanosine triphosphate (GTP) around chromosomes induces local nucleation and plus end stabilization of microtubules (MTs). The nuclear protein TPX2 is required for RanGTP-dependent MT nucleation. To find the MT stabilizer, we affinity purify nuclear localization signal (NLS)–containing proteins from Xenopus laevis egg extracts. This NLS protein fraction contains the MT stabilization activity. After further purification, we used mass spectrometry to identify proteins in active fractions, including cyclin-dependent kinase 11 (Cdk11). Cdk11 localizes on spindle poles and MTs in Xenopus culture cells and egg extracts. Recombinant Cdk11 demonstrates RanGTP-dependent MT stabilization activity, whereas a kinase-dead mutant does not. Inactivation of Cdk11 in egg extracts blocks RanGTP-dependent MT stabilization and dramatically decreases the spindle assembly rate. Simultaneous depletion of TPX2 completely inhibits centrosome-dependent spindle assembly. Our results indicate that Cdk11 is responsible for RanGTP-dependent MT stabilization around chromosomes and that this local stabilization is essential for normal rates of spindle assembly and spindle function.

The nucleoporin MEL-28 promotes RanGTP-dependent γ-tubulin recruitment and microtubule nucleation in mitotic spindle formation

“…CSF extracts were supplemented with Cy3-labelled tubulin and incubated with sperm nuclei or chromatin beads for spindle assembly 30 . Microtubule density around sperm or beads was quantified using Matlab (The MathWorks) 31 . For rescue experiments, mRNA encoding Xenopus MEL-28 (GenBank accession KJ026765) was prepared using the mMESSAGE mMachine kit (Life Technologies) and added to extracts at a concentration of 300 ng ml À 1 .…”

“…His, GFP-tagged aa 1,441-2,201 was constructed in pET28a (Novagen), expressed in E. coli and purified with Ni-NTA and Superose 6 (GE Healthcare Life Sciences). His-importin-a, His-importin-b and His-RanQ69L-GTP were expressed in E. coli and purified with TALON beads (Clontech) 31 .…”

The cyclin-dependent kinase PITSLRE/CDK11 is required for successful autophagy

“…[14][15][16][17][18][19] It is important therefore not to simply focus on how CDK11 regulates components of the autophagy machinery to bring about the effects we report knocked down by siRNA (Fig. 3A).…”

“…13 Subsequent studies on PITSLRE/CDK11 have revealed diverse roles for the kinase in controlling RNA splicing, apoptosis, sister chromatid adhesion and cytokinesis. [14][15][16][17][18][19] We add to these roles by showing that CDK11 is a fundamental regulator of autophagy. We show that while CDK11 appears to act as a turnover.…”

The Biochemistry of Mitosis

“…As a result of nuclear envelope breakdown (NEBD), another Ran-GTP gradient is generated around the chromosomes, to which the RCC1 GTP-exchange factor binds (Clarke 2008). This Ran-GTP gradient is important for the interaction between microtubules and chromosomes because the high Ran-GTP levels around chromosomes promote the dissociation between the importin b chaperone and its binding partners, several of which help to stabilize or nucleate microtubules (Carazo-Salas et al 1999;Kalab et al 1999;Gruss et al 2001;Wilde et al 2001;Yokoyama et al 2008).…”