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A Mitotic Cascade of Nima Family Kinases Nercc1/Nek9 Activates the Nek6 and Nek7 Kinases

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A Mitotic Cascade of Nima Family Kinases Nercc1/Nek9 Activates the Nek6 and Nek7 Kinases A Mitotic Cascade of Nima Family Kinases Nercc1/nek9 Activates the Nek6 and Nek7 Kinases The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Belham, Christopher, Joan Roig, Jennifer A. Caldwell, Yumi Aoyama, Bruce E. Kemp, Michael Comb, and Joseph Avruch. 2003. “A Mitotic Cascade of NIMA Family Kinases.” Journal of Biological Chemistry 278 (37): 34897–909. https://doi.org/10.1074/jbc.m303663200. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:41483059 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 37, Issue of September 12, pp. 34897–34909, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. A Mitotic Cascade of NIMA Family Kinases Nercc1/Nek9 ACTIVATES THE Nek6 AND Nek7 KINASES* Received for publication, April 8, 2003, and in revised form, June 29, 2003 Published, JBC Papers in Press, July 2, 2003, DOI 10.1074/jbc.M303663200 Christopher Belham‡§¶, Joan Roig‡¶ʈ, Jennifer A. Caldwell**, Yumi Aoyama‡, Bruce E. Kemp‡‡§§, Michael Comb¶¶, and Joseph Avruch‡ʈʈ From the ‡Department of Molecular Biology and Medical Services, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114, §Pharmacia Italia S.p.A./Biology Department, 20014 Milan, Italy, **MDS Proteomics, Charlottesville, Virginia 22911, ‡‡St. Vincent’s Institute of Medical Research, Fitzroy, Victoria 3065, Australia, and ¶¶Cell Signaling Technologies, Beverly, Massachusetts 01915 The Nek family of protein kinases in humans is com- breakdown in mammalian cells as it does in Aspergillus (3, 5, posed of 11 members that share an amino-terminal cat- 6), suggesting that these functions are also regulated by pro- alytic domain related to NIMA, an Aspergillus kinase tein kinases with similar specificity in vertebrate cells. Eleven involved in the control of several aspects of mitosis, and protein kinases with a catalytic domain related to NIMA have divergent carboxyl-terminal tails of varying length. been identified in the human genome (7), and a substantial Nek6 (314AA) and Nek7 (303AA), 76% identical, have lit- fraction were first described very recently (8–12). The func- Downloaded from tle noncatalytic sequence but bind to the carboxyl-ter- tions of these NIMA family kinases, mostly referred to as Neks, minal noncatalytic tail of Nercc1/Nek9, a NIMA family are largely unknown. The best characterized of these kinases, protein kinase that is activated in mitosis. Microinjec- Nek2, has been implicated in the regulation of the centrosome tion of anti-Nercc1 antibodies leads to spindle abnor- (13); Nek1 and Nek8 mutations have been related to cystic malities and prometaphase arrest or chromosome mis- kidney disease (14, 15); Nek6/7 have been suggested to phos- segregation. Herein we show that Nek6 is increased in phorylate and activate p70 S6 kinase (16); and Nek9/Nercc1 http://www.jbc.org/ abundance and activity during mitosis; activation re- quires the phosphorylation of Ser206 on the Nek6 activa- has been implicated in the control of mitotic spindle formation tion loop. This phosphorylation and the activity of re- and chromosome segregation (10). combinant Nek6 is stimulated by coexpression with an Nek6 together with its close homolog, Nek7, were purified activated mutant of Nercc1. Moreover, Nercc1 catalyzes from rat liver as the predominant kinases capable of phospho- the direct phosphorylation of prokaryotic recombinant rylating in vitro the hydrophobic regulatory site (Thr412)ofthe Nek6 at Ser206 in vitro concomitant with 20–25-fold ac- p70 S6 kinase in vitro (16). Recombinant Nek6 polypeptide is by guest on October 4, 2019 tivation of Nek6 activity; Nercc1 activates Nek7 in vitro recovered as an active protein kinase after transient expression in a similar manner. Nercc1/Nek9 is likely to be respon- in HEK293 cells and activates coexpressed p70 S6 kinase in sible for the activation of Nek6 during mitosis and prob- vivo (as well as directly in vitro) in a manner synergistic with ably participates in the regulation of Nek7 as well. These PDK1; nevertheless, recent evidence indicates that Nek6 (and findings support the conclusion that Nercc1/Nek9 and by extension Nek7) is not a physiologic activator of p70 S6 Nek6 represent a novel cascade of mitotic NIMA family kinase (17), and thus its roles in cell regulation are unknown. protein kinases whose combined function is important To gain insight into Nek6/7 regulation and function, we for mitotic progression. sought to uncover the mechanism underlying the activation of the Nek6/7 kinases and to define the regulation of the endog- enous enzymes. We find that Nercc1, another NIMA family The NIMA family of protein kinases is named after the kinase previously shown to bind Nek6, phosphorylates directly Aspergillus nidulans protein kinase encoded by the nimA gene a critical site on the activation loop of both Nek6 and Nek7 and (1). Mutation of nimA (never in mitosis A) arrests cells in G2 cdc2 activates these kinases in vitro and in vivo. Moreover, like without interfering with p34 activation (2), suggesting that Nercc1, endogenous Nek6 is activated in mitosis. The ability of the NIMA protein has a central role in the G2/M transition. Nercc1 to directly activate Nek6 points to the likely operation Moreover, if the G2 arrest of nimA mutants is bypassed by of a cascade of NIMA-related mitotic protein kinases. additional mutations, the resulting mitotic cells show aberrant spindle and nuclear envelope organization (3, 4), pointing to EXPERIMENTAL PROCEDURES functions of NIMA beyond the control of mitotic entry. NIMA Materials—Expand HiFidelity DNA polymerase, sequencing grade can induce chromatin condensation and nuclear membrane trypsin, and CompleteTM protease inhibitor mixture tablets were pur- chased from Roche Applied Science. Protein A- and G-Sepharose and GSH-Sepharose were from Amersham Biosciences. Insulin, rapamycin, * This work was supported in part by National Institutes of Health wortmannin, nocodazole, and FLAG M2 antibody were from Sigma. Grant DK17776. The costs of publication of this article were defrayed in Anti-Myc (9E10) monoclonal antibody, anti-cyclin B1 and rabbit anti- part by the payment of page charges. This article must therefore be p70 polyclonal antibody (C-18) were purchased from Santa Cruz Bio- hereby marked “advertisement” in accordance with 18 U.S.C. Section technology, Inc. (Santa Cruz, CA). Anti-␤-tubulin was from Zymed 1734 solely to indicate this fact. ¶ These two authors contributed equally to this work. Laboratories Inc.. LipofectAMINE, pcDNA3.1-Myc/His6 mammalian ʈ Supported in part by the Fund for Medical Discovery from Massa- expression vector, and all cell culture media except phosphate-free chusetts General Hospital and the Leukemia and Lymphoma Society. Dulbecco’s modified Eagle’s medium (ICN) was from Invitrogen. Cellu- §§ A National Health and Medical Research Council Fellow and sup- lose TLC plates were bought from E.M. Science. Rabbit polyclonal sera ported by the Australian Research Council. raised against keyhole limpet hemocyanin-coupled peptide were gener- ʈʈ To whom correspondence should be addressed. E-mail: avruch@ ated at Cocalico Biologicals (Reamstown, PA). [32P]orthophosphate and helix.mgh.harvard.edu. [␥-32P]ATP were obtained from PerkinElmer Life Sciences and ICN, This paper is available on line at http://www.jbc.org 34897 34898 Nercc1/Nek9 Activates Nek6 and Nek7 respectively. MBP1 was purchased from Upstate Biotechnology, Inc. were manually confirmed to ensure correct sequence identification. (Lake Placid, NY). HEK293, H4-II-E-C3, HeLa, and U2OS cells were Nek6 Kinase Assay—Immunoprecipitates of recombinant (anti- obtained from ATCC. FLAG or anti-Myc) or endogenous Nek6 were first washed in the ex- Construction of Expression Plasmids—The mammalian expression traction buffer containing 0.5 M LiCl three times and twice in kinase vectors PEBG 2T and pCMV5 FLAG encoding wild type and kinase- buffer (50 mM MOPS, pH 7.4, 10 mM MgCl2,2mM EGTA, 20 mM dead (K74M/K75M) Nek6, the pCMV5 FLAG plasmid encoding differ- ␤-glycerophosphate). Kinase assays were performed in 30 ␮l of kinase ent forms of Nercc1 and the bacterial expression vector pGEX KG buffer containing 1.5 ␮g of GST-p70 S6 kinase ⌬CT104 (T252A) or MBP expressing GST-Nercc1-(732–979) have been described previously (10, (0.2 mg/ml) and initiated by the addition of [␥-32P]ATP (5 ␮M final 16, 18). For construction of pcDNA3.1 His6/Myc Nek6, a PCR fragment concentration at 4000 cpm/pmol). Reactions were incubated for 10 min was generated using pCMV5 FLAG Nek6 as template encoding the at 30 °C and terminated by the addition of 4ϫ SDS sample buffer. open reading frame of human Nek6 lacking the stop codon and sur- Kinase mixtures were resolved by SDS-PAGE and transferred to poly- rounded at the initiator methionine by an optimum Kozak sequence. vinylidene difluoride membrane, and Nek6 phosphotransferase activity This fragment was subcloned into pcDNA3.1 Myc/His6, allowing Nek6 was determined by excising Coomassie-stained bands corresponding to to be expressed in mammalian cells as a fusion protein with His6 and the substrate and measuring radioactive content by Cerenkov scintil- Myc epitopes at the C terminus. lation counting. All site-directed mutant Nek6 variants in pcDNA3.1 Myc/His6 were Two-dimensional Tryptic Phosphopeptide Mapping—HEK293 cells constructed by PCR-mediated overlap extension mutagenesis and sub- expressing various Nek6-His6/Myc constructs were incubated with 32 sequently subcloned into pCDNA3.1 Myc/His6. All clones were verified phosphate-free Dulbecco’s modified Eagle’s medium containing Pi for by sequencing. 4 h prior to harvest. The Myc immunoprecipitates were subjected to Generation of Rabbit Anti-Nek6 Antisera—A synthetic peptide corre- SDS-PAGE, fixation, and staining.
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