The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores organized from 2 centrosomes (a.k.a. spindle poles). In addition to this central spindle apparatus, astral MTs assemble at the mitotic spindle pole and attach to the cell cortex to ensure appropriate spindle orientation. We propose that cell cycle-related kinase, Nek7, and its novel interacting protein RGS2, are involved in mitosis regulation and spindle formation. We found that RGS2 localizes to the mitotic spindle in a Nek7-dependent manner, and along with Nek7 contributes to spindle morphology and mitotic spindle pole integrity. RGS2-depletion leads to a mitotic-delay and severe defects in the chromosomes alignment and congression. Importantly, RGS2 or Nek7 depletion or even overexpression of wild-type or kinase-dead Nek7, reduced γ-tubulin from the mitotic spindle poles. In addition to causing a mitotic delay, RGS2 depletion induced mitotic spindle misorientation coinciding with astral MT-reduction. We propose that these phenotypes directly contribute to a failure in mitotic spindle alignment to the substratum. In conclusion, we suggest a molecular mechanism whereupon Nek7 and RGS2 may act cooperatively to ensure proper mitotic spindle organization.144656667Goshima, G., Wollman, R., Goodwin, S.S., Zhang, N., Scholey, J.M., Vale, R.D., Stuurman, N., Genes required for mitotic spindle assembly in Drosophila S2 cells (2007) Science, 5823, pp. 417-421. , http://dx.doi.org/10.1126/science.1141314Uehara, R., Nozawa, R.S., Tomioka, A., Petry, S., Vale, R.D., Obuse, C., Goshima, G., The augmin complex plays a critical role in spindle microtubule generation for mitotic progression and cytokinesis in human cells (2009) Proc Natl Acad Sci USA, 106, pp. 6998-7003. , http://dx.doi.org/10.1073/pnas.0901587106, PMID:19369198Dumont, S., Mitchison, T.J., Force and length in the mitotic spindle (2009) Curr Biol, 17, pp. R749-R761. , http://dx.doi.org/10.1016/j.cub.2009.07.028Hayward, D., Metz, J., Pellacani, C., Wakefield, J.G., Synergy between multiple microtubule-generating pathways confers robustness to centrosome-driven mitotic spindle formation (2014) Dev Cell, 1, pp. 81-93. , http://dx.doi.org/10.1016/j.devcel.2013.12.001Luders, J., Stearns, T., Microtubule-organizing centres: A re-evaluation (2007) Nat Rev Mol Cell Biol, 8, pp. 161-167. , http://dx.doi.org/10.1038/nrm2100, PMID:17245416Willard, F.S., Kimple, R.J., Siderovski, D.P., Return of the GDI: The GoLoco motif in cell division (2004) Annu Rev Biochem, 73, pp. 925-951. , http://dx.doi.org/10.1146/annurev.biochem.73.011303.073756, PMID:15189163Kotak, S., Gönczy, P., Mechanisms of spindle positioning: Cortical force generators in the limelight (2013) Curr Opin Cell Biol, 6, pp. 741-748. , http://dx.doi.org/10.1016/j.ceb.2013.07.008Zheng, Z., Wan, Q., Liu, J., Zhu, H., Chu, X., Du, Q., Evidence for dynein and astral microtubule-mediated cortical release and transport of Gai/LGN/NuMA complex in mitotic cells (2013) Mol Biol Cell, 7, pp. 901-913. , http://dx.doi.org/10.1091/mbc.E12-06-0458Musacchio, A., Salmon, E.D., The spindle-assembly checkpoint in space and time (2007) Nat Rev Mol Cell Biol, 8, pp. 379-393. , http://dx.doi.org/10.1038/nrm2163, PMID:17426725Noatynska, A., Gotta, M., Meraldi, P., Mitotic spindle (DIS) orientation and DISease: Cause or consequence? (2012) J Cell Biol, 7, pp. 1025-1035. , http://dx.doi.org/10.1083/jcb.201209015O'Connell, M.J., Krien, M.J., Hunter, T., Never say never. The NIMA-related protein kinases in mitotic control (2003) Trends Cell Biol, 5, pp. 221-228. , http://dx.doi.org/10.1016/S0962-8924(03)00056-4O'Regan, L., Blot, J., Fry, A.M., Mitotic regulation by NIMA-related kinases (2007) Cell Div, 29, pp. 2-25Fry, A.M., O'Regan, L., Sabir, S.R., Bayliss, R., Cell cycle regulation by the NEK family of protein kinases (2012) J Cell Sci, 125, pp. 4423-4433. , http://dx.doi.org/10.1242/jcs.111195, PMID:23132929Meirelles, G.V., Perez, A.M., De Souza, E.E., Basei, F.L., Papa, P.F., Hanchuk, T.D.M., Cardoso, V.B., Kobarg, J., "Stop Ne (c)king around:" How systems biology can help to characterize the functions of NEK family kinases from cell cycle regulation to DNA damage response (2014) World J Biol Chem, 5 (2), pp. 141-160Roig, J., Groen, A., Caldwell, J., Avruch, J., Active Nercc1 protein kinase concentrates at centrosomes early in mitosis and is necessary for proper spindle assembly (2005) Mol Biol Cell, 16, pp. 4827-4840. , http://dx.doi.org/10.1091/mbc.E05-04-0315, PMID:16079175Yissachar, N., Salem, H., Tennenbaum, T., Motro, B., Nek7 kinase is enriched at the centrosome, and is required for proper spindle assembly and mitotic progression (2006) FEBS Lett, 27, pp. 6489-6495. , http://dx.doi.org/10.1016/j.febslet.2006.10.069Kim, S., Lee, K., Rhee, K., NEK7 is a centrosomal kinase critical for microtubule nucleation (2007) Biochem Biophys Res Commun, 1, pp. 56-62. , http://dx.doi.org/10.1016/j.bbrc.2007.05.206O'Regan, L., Fry, A.M., The Nek6 and Nek7 protein kinases are required for robust mitotic spindle formation and cytokinesis (2009) Mol Cell Biol, 14, pp. 3975-3990. , http://dx.doi.org/10.1128/MCB.01867-08Belham, C., Roig, J., Caldwell, J.A., Aoyama, Y., Kemp, B.E., Comb, M., Avruch, J.A., Mitotic cascade of NIMA family kinases. Nercc1/NEK9 activates the NEK6 and NEK7 kinases (2003) J Biol Chem, 37, pp. 34897-34909. , http://dx.doi.org/10.1074/jbc.M303663200Richards, M.W., O'Regan, L., Mas-Droux, C., Blot, J.M., Cheung, J., Hoelder, S., Fry, A.M., Bayliss, R., Anautoinhibitory tyrosine motif in the cell-cycle-regulated NEK7 kinase is released through binding of NEK9 (2009) Mol Cell, 4, pp. 560-570. , http://dx.doi.org/10.1016/j.molcel.2009.09.038Quarmby, L.M., Mahjoub, M.R., Caught Nek-ing: Cilia and centrioles (2005) J Cell Sci, 118, pp. 5161-5169. , http://dx.doi.org/10.1242/jcs.02681, PMID:16280549Kim, S., Rhee, K., NEK7 is essential for centriole duplication and centrosomal accumulation of pericentriolar material proteins in interphase cells (2011) J Cell Sci, 124, pp. 3760-3770. , http://dx.doi.org/10.1242/jcs.078089, PMID:22100915Salem, H., Rachmin, I., Yissachar, N., Cohen, S., Amiel, A., Haffner, R., Lavi, L., Motro, B., Nek7 kinase targeting leads to early mortality, cytokinesis disturbance and polyploidy (2010) Oncogene, 28, pp. 4046-4057. , http://dx.doi.org/10.1038/onc.2010.162De Souza, E.E., Meirelles, G.V., Godoy, B.B., Perez, A.M., Smetana, J.H., Doxsey, S.J., McComb, M.E., Kobarg, J., Characterization of the human NEK7 interactome suggests catalytic and regulatory properties distinct from those of NEK6 (2014) J Proteome Res, 13 (9), pp. 4074-4090. , http://dx.doi.org/10.1021/pr500437x, PMID:25093993Abramow-NewerlyM, Roy, A.A., Nunn, C., Chidiac, P., RGS proteins have a signalling complex: Interactions between RGS proteins and GPCRs, effectors, and auxiliary proteins (2006) Cell Signal, 18, pp. 579-591. , http://dx.doi.org/10.1016/j.cellsig.2005.08.010, PMID:16226429Bastin, G., Heximer, S.P., Rab family proteins regulate the endosomal trafficking and function of RGS4 (2013) J Biol Chem., 30, pp. 21836-21849. , http://dx.doi.org/10.1074/jbc.M113.466888Siderovski, D.P., Hessel, A., Chung, S., Mak, T.W., Tyers, M., A new family of regulators of G-protein-coupled receptors? (1996) Curr Biol, 2, pp. 211-212. , http://dx.doi.org/10.1016/S0960-9822(02)00454-2Keys, J.R., Greene, E.A., Koch, W.J., Eckhart, A.D., Gq-coupled receptor agonists mediate cardiac hypertrophy via the vasculature (2002) Hypertension, 40, pp. 660-666. , http://dx.doi.org/10.1161/01.HYP.0000035397.73223.CE, PMID:12411459Wilkie, T.M., Kinch, L., New roles for G a and RGS proteins: Communication continues despite pulling sisters apart (2005) Curr Biol, 15, pp. 843-854. , http://dx.doi.org/10.1016/j.cub.2005.10.008Hewavitharana, T., Wedegaertner, P.B., Non-canonical signaling and localizations of heterotrimeric G proteins (2012) Cell Signal, 1, pp. 25-34. , http://dx.doi.org/10.1016/j.cellsig.2011.08.014Lampson, M.A., Cheeseman, I.M., Sensing centromere tension: Aurora B and the regulation of kinetochore function (2011) Trends Cell Biol, 3, pp. 133-140. , http://dx.doi.org/10.1016/j.tcb.2010.10.007Hochegger, H., H'Egarat, N., Pereira-Leal, J.B., Aurora at the pole and equator: Overlapping functions of Aurora kinases in the mitotic spindle (2013) Open Biol, 3, p. 120185. , http://dx.doi.org/10.1098/rsob.120185, PMID:23516109Hehnly, H., Doxsey, S., Rab11 endosomes contribute to mitotic spindle organization and orientation (2014) Dev Cell, 28, pp. 497-507. , http://dx.doi.org/10.1016/j.devcel.2014.01.014, PMID:24561039Cowley, D.O., Rivera-P'Erez, J.A., Schliekelman, M., He, Y.J., Oliver, T.G., Lu, L., O'Quinn, R., Van Dyke, T., Aurora-A kinase is essential for bipolar spindle formation and early development (2009) Mol Cell Biol, 4, pp. 1059-1071. , http://dx.doi.org/10.1128/MCB.01062-08Nagai, T., Ikeda, M., Chiba, S., Kanno, S., Mizuno, K., Furry promotes acetylation of microtubules in the mitotic spindle by inhibition of SIRT2 tubulin deacetylase (2013) J Cell Sci, 19, pp. 4369-4380. , http://dx.doi.org/10.1242/jcs.127209Zimmerman, W.C., Sillibourne, J., Rosa, J., Doxsey, S.J., Mitosis-specific anchoring of gamma tubulin complexes by pericentrin controls spindle organization and mitotic entry (2004) Mol Biol Cell, 8, pp. 3642-3657. , http://dx.doi.org/10.1091/mbc.E03-11-0796Bornens, M., The centrosome in cells and organisms (2012) Science, 335, pp. 422-426. , http://dx.doi.org/10.1126/science.1209037, PMID:22282802Bouissou, A., V'Erollet, C., De Forges, H., Haren, L., Bellä Iche, Y., Perez, F., Merdes, A., Raynaud-Messina, B., γ-Tubulin ring complexes and EB1 play antagonistic roles in microtubule dynamics and spindle positioning (2014) EMBO J, 2, pp. 114-128. , http://dx.doi.org/10.1002/embj.201385967Laan, L., Pavin, N., Husson, J., Romet-Lemonne, G., Van Duijn, M., Ĺopez, M.P., Vale, R.D., Dogterom, M., Cortical dynein controls microtubule dynamics to generate pulling forces that position microtubule asters (2012) Cell, 3, pp. 502-514. , http://dx.doi.org/10.1016/j.cell.2012.01.007Delaval, B., Bright, A., Lawson, N.D., Doxsey, S., The cilia protein IFT88 is required for spindle orientation in mitosis (2011) Nat Cell Biol, 4, pp. 461-468. , http://dx.doi.org/10.1038/ncb2202Lu, M.S., Johnston, C.A., Molecular pathways regulating mitotic spindle orientation in animal cells (2013) Development, 9, pp. 1843-1856. , http://dx.doi.org/10.1242/dev.087627Cohen, S., Aizer, A., Shav-Tal, Y., Yanai, A., Motro, B., Nek7 kinase acceleratesmicrotubule dynamic instability (2013) Biochim Biophys Acta, 1833, pp. 1104-1113. , http://dx.doi.org/10.1016/j.bbamcr.2012.12.021, PMID:23313050Welburn, J.P., Vleugel, M., Liu, D., Yates, J.R., Lampson, M.A., Fukagawa, T., Cheeseman, I.M., Aurora B phosphorylates spatially distinct targets to differentially regulate the kinetochore-microtubule interface (2010) Mol Cell, 38 (3), pp. 383-392. , http://dx.doi.org/10.1016/j.molcel.2010.02.034, PMID:20471944Heo, K., Ha, S.H., Chae, Y.C., Lee, S., Oh, Y.S., Kim, Y.H., Kim, S.H., Ryu, S.H., Suh PG RGS2 promotes formation of neurites by stimulating microtubule polymerization (2006) Cell Signal, 12, pp. 2182-2192. , http://dx.doi.org/10.1016/j.cellsig.2006.05.006Blumer, J.B., Kuriyama, R., Gettys, T.W., Lanier, S.M., The G-protein regulatory (GPR) motif-containing Leu-Gly-Asn-enriched protein (LGN) and Gialpha3 influence cortical positioning of the mitotic spindle poles at metaphase in symmetrically dividing mammalian cells (2006) Eur J Cell Biol, 12, pp. 1233-1240. , http://dx.doi.org/10.1016/j.ejcb.2006.08.002Woodard, G.E., Huang, N.-N., Cho, H., Miki, T., Tall, G.G., Kehrl, J.H., Ric-8A and Gi Alpha recruit LGN, NuMA, and dynein to the cell cortex to help orient the mitotic spindle (2010) Mol Cell Biol, 14, pp. 3519-3530. , http://dx.doi.org/10.1128/MCB.00394-10Upadhya, P., Birkenmeier, E.H., Birkenmeier, C.S., Barker, J.E., Mutations in a NIMA-related kinase gene, Nek1, cause peliotropic effects including a progressive polycystic kidney disease in mice (2000) Proc Natl Acad Sci U S A, 97, pp. 217-221. , http://dx.doi.org/10.1073/pnas.97.1.217, PMID:10618398Zalli, D., Bayliss, R., Fry, A.M., The Nek8 protein kinase, mutated in the human cystic kidney disease nephronophthisis, is both activated and degraded during ciliogenesis (2012) Hum Mol Genet, 5, pp. 1155-1171. , http://dx.doi.org/10.1093/hmg/ddr544Moniz, L., Dutt, P., Haider, N., Stambolic, V., Nek family of kinases in cell cycle, checkpoint control and cancer (2011) Cell Div, 1, p. 18. , http://dx.doi.org/10.1186/1747-1028-6-18Kawamura, E., Fielding, A.B., Kannan, N., Balgi, A., Eaves, C.J., Roberge, M., Dedhar, S., Identification of novel small molecule inhibitors of centrosome clustering in cancer cells (2013) Oncotarget, 4, pp. 1763-1776. , PMID:24091544Sanhaji, M., Ritter, A., Belsham, H.R., Friel, C.T., Roth, S., Louwen, F., Yuan, J., Polo-like kinase 1 regulates the stability of themitotic centromere-associated kinesin inmitosis (2014) Oncotarget, 5, pp. 3130-3144. , PMID:24931513Carazzolle, M.F., De Carvalho, L.M., Slepicka, H.H., Vidal, R.O., Pereira, G.A., Kobarg, J., Meirelles, G.V., IIS - Integrated Interactome System: A web-based platform for the annotation, analysis and visualization of protein-metabolite-gene-drug interactions by integrating a variety of data sources and tools (2014) PLoS One, 9 (6), p. e100385. , http://dx.doi.org/10.1371/journal.pone.0100385Shannon, P., Markiel, A., Ozier, O., Baliga, N.S., Wang, J.T., Ramage, D., Amin, N., Ideker, T., Cytoscape: A software environment for integrated models of biomolecular interaction networks (2003) Genome Res, 11, pp. 2498-2504. , http://dx.doi.org/10.1101/gr.1239303Thoma, C.R., Toso, A., Gutbrodt, K.L., Reggi, S.P., Frew, I.J., Schraml, P., Hergovich, A., Krek, W., VHL loss causes spindle misorientation and chromosome instability (2009) Nat Cell Biol, 8, pp. 994-1001. , http://dx.doi.org/10.1038/ncb1912