Characterization Of The Human Nek7 Interactome Suggests Catalytic And Regulatory Properties Distinct From Those Of Nek6

Human NEK7 is a regulator of cell division and plays an important role in growth and survival of mammalian cells. Human NEK6 and NEK7 are closely related, consisting of a conserved C-terminal catalytic domain and a nonconserved and disordered N-terminal regulatory domain, crucial to mediate the interactions with their respective proteins. Here, in order to better understand NEK7 cellular functions, we characterize the NEK7 interactome by two screening approaches: one using a yeast two-hybrid system and the other based on immunoprecipitation followed by mass spectrometry analysis. These approaches led to the identification of 61 NEK7 interactors that contribute to a variety of biological processes, including cell division. Combining additional interaction and phosphorylation assays from yeast two-hybrid screens, we validated CC2D1A, TUBB2B, MNAT1, and NEK9 proteins as potential NEK7 interactors and substrates. Notably, endogenous RGS2, TUBB, MNAT1, NEK9, and PLEKHA8 localized with NEK7 at key sites throughout the cell cycle, especially during mitosis and cytokinesis. Furthermore, we obtained evidence that the closely related kinases NEK6 and NEK7 do not share common interactors, with the exception of NEK9, and display different modes of protein interaction, depending on their N- and C-terminal regions, in distinct fashions. In summary, our work shows for the first time a comprehensive NEK7 interactome that, combined with functional in vitro and in vivo assays, suggests that NEK7 is a multifunctional kinase acting in different cellular processes in concert with cell division signaling and independently of NEK6.13940744090Ma, H.T., Poon, R.Y., How protein kinases co-ordinate mitosis in animal cells (2011) Biochem. J., 435, pp. 17-31Fry, A.M., Meraldi, P., Nigg, E.A., A centrosomal function for the human NEK2 protein kinase, a member of the NIMA family of cell cycle regulators (1998) EMBO J., 17, pp. 470-481Moniz, L., Dutt, P., Haider, N., Stambolic, V., NEK family of kinases in cell cycle, checkpoint control and cancer (2011) Cell Div., 6, p. 18Dodson, C.A., Haq, T., Yeoh, S., Fry, A.M., Bayliss, R., The structural mechanisms that underpin mitotic kinase activation (2013) Biochem. Soc. Trans., 41, pp. 1037-1041Morris, N.R., Mitotic mutants of Aspergillus nidulans (1975) Genet. Res., 26, pp. 237-254Oakley, B.R., Morris, N.R., A mutation in Aspergillus nidulans that blocks the transition from interphase to prophase (1983) J. Cell Biol., 96, pp. 1155-1158Belham, 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., 278, pp. 34897-34909Li, J.J., Li, S.A., Mitotic kinases: The key to duplication, segregation, and cytokinesis errors, chromosomal instability, and oncogenesis (2006) Pharmacol Ther., 111, pp. 974-984Upadhya, P., Birkenmeier, E.H., Birkenmeier, C.S., Barker, J.E., Mutations in a NIMA-related kinase gene, NEK1, cause pleiotropic effects including a progressive polycystic kidney disease in mice (2000) Proc. Natl. Acad. Sci. U.S.A., 97, pp. 217-221Quarmby, L.M., Mahjoub, M.R., Caught NEK-ing: Cilia and centrioles (2005) J. Cell Sci., 118, pp. 5161-5169Kim, S., Lee, K., Rhee, K., NEK7 is a centrosomal kinase critical for microtubule nucleation (2007) Biochem. Biophys. Res. Commun., 360, pp. 56-62O'Regan, L., Blot, J., Fry, A.M., Mitotic regulation by NIMA-related kinases (2007) Cell Div., 2, p. 25O'Regan, L., Fry, A.M., The NEK6 and NEK7 protein kinases are required for robust mitotic spindle formation and cytokinesis (2009) Mol. Cell. Biol., 29, pp. 3975-3990Yissachar, 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., 580, pp. 6489-6495Kim, S., Kim, 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. 3760-3770Salem, 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, 29, pp. 4046-4057Kandli, M., Feige, E., Chen, A., Kilfin, G., Motro, B., Isolation and characterization of two evolutionarily conserved murin kinases (NEK6 and NEK7) related to the fungal mitotic regulator, NIMA (2000) Genomics, 68, pp. 187-196Richards, M.W., O'Regan, L., Mas-Droux, C., Blot, J.M., Cheung, J., Hoelder, S., Fry, A.M., Bayliss, R., An autoinhibitory tyrosine motif in the cell-cycle-regulated NEK7 kinase is released through binding of NEK9 (2009) Mol. Cell, 36, pp. 560-570Meirelles, G.V., Silva, J.C., Mendonça Yde, A., Ramos, C.H., Torriani, I.L., Kobarg, J., Human NEK6 is a monomeric mostly globular kinase with an unfolded short N-terminal domain (2011) BMC Struct. Biol., 11, p. 12Feige, E., Motro, B., The related murine kinases, NEK6 and NEK7, display distinct patterns of expression (2002) Mech. Dev., 110, pp. 219-223Minoguchi, S., Minoguchi, M., Yoshimura, A., Differential control of the NIMA related kinases, NEK6 and NEK7, by serum stimulation (2003) Biochem. Biophys. Res. Commun., 301, pp. 899-906Meirelles, G.V., Lanza, D.C.F., Silva, J.C., Bernachi, J.S., Leme, A.F.P., Kobarg, J., Characterization of NEK6 interactome reveals an important role for its short n-terminal domain and colocalization with proteins at the centrosome (2010) J. Proteome Res., 9, pp. 6298-6316Bartel, P.L., Fields, S., Analyzing protein-protein interactions using two-hybrid system (1995) Methods Enzymol., 254, pp. 241-263Devault, A., Martinez, A.M., Fesquet, D., Labbe, J.C., Morin, N., Tassano, J.P., Nigg, E.A., Doree, M., MNAT1 ("ménage à trois') a new RING finger protein subunit stabilizing ciclin H-cdk7 complexes in starfish and Xenopus CAK (1995) EMBO J., 14, pp. 5027-5036Whelan, S.A., He, J., Lu, M., Souda, P., Saxton, R.E., Faull, K.F., Whitelegge, J.P., Chang, H.R., Mass spectrometry (LC-MS/MS) identified proteomic biosignatures of breast cancer in proximal fluid (2012) J. Proteome Res., 11, pp. 5034-5045Carazzolle, 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, p. 100385Shannon, 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., 13, pp. 2498-2504Surpili, M.J., Delben, T.M., Kobarg, J., Identification of proteins that interact with the central coiled-Ccoil region of the human protein kinase NEK1 (2003) Biochemistry, 42, pp. 15369-15376Bartel, P., Chien, C., Sternglanz, R., Fields, S., (1993) Cellular Interactions in Development: A Practical Approach, pp. 153-179. , Hartley, D.A. Oxford University Press: OxfordPark, S., Lim, B.B., Perez-Terzic, C., Mer, G., Terzic, A., Interaction of asymmetric ABCC9-encoded nucleotide binding domains determines KATP channel SUR2A catalytic activity (2008) J. Proteome Res., 7, pp. 1721-1728Hegde, M.L., Hazra, T.K., Mitra, S., Functions of disordered regions in mammalian early base excision repair proteins (2010) Cell. Mol. Life Sci., 67, pp. 3573-3587Mittag, T., Kay, L.E., Forman-Kay, J.D., Protein dynamics and conformational disorder in molecular recognition (2010) J. Mol. Recognit., 23, pp. 105-116Doxsey, S.J., Stein, P., Evans, L., Calarco, P.D., Kirschner, M., Pericentrin highly conserved centrosome protein involved in microtubule organization (1994) Cell, 76, pp. 639-650Belham, C., Comb, M.J., Avruch, J., Identification of the NIMA family kinases NEK6/7 as regulators of the p70 ribosomal S6 kinase (2011) Curr. Biol., 11, pp. 1155-1167Rapley, J., Nicolàs, M., Groen, A., Regué, L., Bertran, M.T., Caelles, C., Avruch, J., Roig, J., The NIMA-family kinase NEK6 phosphorylates the kinesin Eg5 at a novel site necessary for mitotic spindle formation (2008) J. Cell Sci., 121, pp. 3912-3921Fourest-Lieuvin, A., Peris, L., Gache, V., Garcia-Saez, I., Juillan-Binard, C., Lantez, V., Job, D., Microtubule regulation in mitosis: Tubulin phosphorylation by the cyclin-dependent kinase Cdk1 (2006) Mol. Biol. Cell, 17, pp. 1041-1050Xu, W., Xi, B., Wu, J., An, H., Zhu, J., Abassi, Y., Feinstein, S.C., Xu, X., Natural product derivative bis(4-fluorobenzyl) trisulfide inhibits tumor growth by modification of beta-tubulin at Cys 12 and suppression of microtubule dynamics (2009) Mol. Cancer Ther., 8, pp. 3318-3330Meirelles, G.V., Perez, A.M., 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, pp. 141-160Ewing, R.M., Chu, P., Elisma, F., Li, H., Taylor, P., Climie, S., McBroom-Cerajewski, L., Figeys, D., Large-scale mapping of human protein-protein interactions by mass spectrometry (2007) Mol. Syst. Biol., 3, p. 8

A Kinesin Mutant with an Atypical Bipolar Spindle Undergoes Normal Mitosis

Motor proteins have been implicated in various aspects of mitosis, including spindle assembly and chromosome segregation. Here, we show that acentrosomal Arabidopsis cells that are mutant for the kinesin, ATK1, lack microtubule accumulation at the predicted spindle poles during prophase and have reduced spindle bipolarity during prometaphase. Nonetheless, all abnormalities are rectified by anaphase and chromosome segregation appears normal. We conclude that ATK1 is required for normal microtubule accumulation at the spindle poles during prophase and possibly functions in spindle assembly during prometaphase. Because aberrant spindle morphology in these mutants is resolved by anaphase, we postulate that mitotic plant cells contain an error-correcting mechanism. Moreover, ATK1 function seems to be dosage-dependent, because cells containing one wild-type allele take significantly longer to proceed to anaphase as compared with cells containing two wild-type alleles

Identification of a Novel Light Intermediate Chain (D2LIC) for Mammalian Cytoplasmic Dynein 2

The diversity of dynein's functions in mammalian cells is a manifestation of both the existence of multiple dynein heavy chain isoforms and an extensive set of associated protein subunits. In this study, we have identified and characterized a novel subunit of the mammalian cytoplasmic dynein 2 complex. The sequence similarity between this 33-kDa subunit and the light intermediate chains (LICs) of cytoplasmic dynein 1 suggests that this protein is a dynein 2 LIC (D2LIC). D2LIC contains a P-loop motif near its NH(2) terminus, and it shares a short region of similarity to the yeast GTPases Spg1p and Tem1p. The D2LIC subunit interacts specifically with DHC2 (or cDhc1b) in both reciprocal immunoprecipitations and sedimentation assays. The expression of D2LIC also mirrors that of DHC2 in a variety of tissues. D2LIC colocalizes with DHC2 at the Golgi apparatus throughout the cell cycle. On brefeldin A-induced Golgi fragmentation, a fraction of D2LIC redistributes to the cytoplasm, leaving behind a subset of D2LIC that is localized around the centrosome. Our results suggest that D2LIC is a bona fide subunit of cytoplasmic dynein 2 that may play a role in maintaining Golgi organization by binding cytoplasmic dynein 2 to its Golgi-associated cargo