KIF14 and citron kinase act together to promote efficient cytokinesis

Abstract

Multiple mitotic kinesins and microtubule-associated proteins (MAPs) act in concert to direct cytokinesis (Glotzer, M. 2005. Science. 307:1735–1739). In anaphase cells, many of these proteins associate with an antiparallel array of microtubules termed the central spindle. The MAP and microtubule-bundling protein PRC1 (protein-regulating cytokinesis 1) is one of the key molecules required for the integrity of this structure (Jiang, W., G. Jimenez, N.J. Wells, T.J. Hope, G.M. Wahl, T. Hunter, and R. Fukunaga. 1998. Mol. Cell. 2:877–885; Mollinari, C., J.P. Kleman, W. Jiang, G. Schoehn, T. Hunter, and R.L. Margolis. 2002. J. Cell Biol. 157:1175–1186). In this study, we identify an interaction between endogenous PRC1 and the previously uncharacterized kinesin KIF14 as well as other mitotic kinesins (MKlp1/CHO1, MKlp2, and KIF4) with known functions in cytokinesis (Hill, E., M. Clarke, and F.A. Barr. 2000. EMBO J. 19:5711–5719; Matuliene, J., and R. Kuriyama. 2002. Mol. Biol. Cell. 13:1832–1845; Kurasawa, Y., W.C. Earnshaw, Y. Mochizuki, N. Dohmae, and K. Todokoro. 2004. EMBO J. 23:3237–3248). We find that KIF14 targets to the central spindle via its interaction with PRC1 and has an essential function in cytokinesis. In KIF14-depleted cells, citron kinase but not other components of the central spindle and cleavage furrow fail to localize. Furthermore, the localization of KIF14 and citron kinase to the central spindle and midbody is codependent, and they form a complex depending on the activation state of citron kinase. Contrary to a previous study (Di Cunto, F., S. Imarisio, E. Hirsch, V. Broccoli, A. Bulfone, A. Migheli, C. Atzori, E. Turco, R. Triolo, G.P. Dotto, et al. 2000. Neuron. 28:115–127), we find a general requirement for citron kinase in human cell division. Together, these findings identify a novel pathway required for efficient cytokinesis.

Tissue-specific control of midbody microtubule stability by Citron kinase through modulation of TUBB3 phosphorylation

“…(b) The same analysis performed in (a) was repeated on HeLa cells treated for 48 h with either control siRNA or with previously validated siRNAs designed on CIT-K mRNA 23 or KIF14. 15 In the case of CIT-K, similar results were obtained using previously validated shRNA-expressing plasmids 23 Statistical significance of differences in average was established using Student's two-tailed t-test. **Po0.01; ***Po0.001…”

“…Previous results indicated that the primary function of CIT-K, conserved from insects to mammals, is to promote the stability and the maturation of midbody before abscission. 15,[23][24][25] To assess whether this function may involve the stabilization of midbody microtubules, we measured the ratio of acetylated versus tyrosinated α-tubulin at the midbody of control cells and of cells lacking CIT-K. 28 Indeed, dynamic microtubules contain a relatively high amount of α-tubulin tyrosinated at its carboxyterminus, whereas stable microtubules show a high content of α-tubulin acetylated at the K40 residue. 29 We studied two cellular models characterized by different sensitivity to CIT-K loss and by different physiological relevance: cultures of proliferating neocortical neuronal progenitors obtained from control or from Cit-K knockout mice at embryonic day (E)12.5 and HeLa cells depleted of CIT-K by RNAi.…”

“…In contrast, the ratio is not significantly different from the control in nondividing progenitors and HeLa cells, and outside of the midbody in telophase HeLa cells (Supplementary Figures S1A and B). We then evaluated whether the altered ratio between tyrosinated and acetylated tubulin produced by CIT-K loss could depend on KIF14, a microtubule-binding protein that has been shown to work immediately downstream of CIT-K in Drosophila 25 and has been proposed to do so also in mammalian cells 15,25 by depleting this protein in cultured wild-type neuronal progenitors and in HeLa cells (Supplementary Figure S2A). However, cells depleted for KIF14 did not show significant differences when compared with the corresponding controls (Figures 2a and b).…”

“…Citron kinase (CIT-K), a conserved ser/thr protein kinase that binds to active RhoA, 13,14 is localized at the cleavage furrow and at the midbody of dividing cells. 13 CIT-K was first considered a 'core' cytokinesis protein because it is ubiquitously expressed in proliferating cells, 13,15 is conserved from insects to mammals [16][17][18][19] and is required by many cultured cell types to complete cytokinesis. 15 However, the characterization of CIT-K knockout mice and the finding of a spontaneous rat mutant have demonstrated that, in vivo and in mammals, CIT-K is not ubiquitously required.…”

“…13 CIT-K was first considered a 'core' cytokinesis protein because it is ubiquitously expressed in proliferating cells, 13,15 is conserved from insects to mammals [16][17][18][19] and is required by many cultured cell types to complete cytokinesis. 15 However, the characterization of CIT-K knockout mice and the finding of a spontaneous rat mutant have demonstrated that, in vivo and in mammals, CIT-K is not ubiquitously required. 20,21 Indeed, CIT-K knockout mice and rats display cytokinesis failure only in few cell types, such as neuronal progenitors 21 and testicular germ cells.…”