The ATPase Cycle of the Mitotic Motor CENP-E

Abstract

We have previously shown that the mitotic motor centrosome protein E (CENP-E) is capable of walking for more than 250 steps on its microtubule track without dissociating. We have examined the kinetics of this molecular motor to see if its enzymology explains this remarkable degree of processivity. We find that like the highly processive transport motor kinesin 1, the enzymatic cycle of CENP-E is characterized by rapid ATP binding, multiple enzymatic turnovers per diffusive encounter, and gating of nucleotide binding. These features endow CENP-E with a high duty cycle, a prerequisite for processivity. However, unlike kinesin 1, neck linker docking in CENP-E is slow, occurring at a rate closer to that for Eg5, a mitotic kinesin that takes only 5–10 steps per processive run. These results suggest that like kinesin 1, features outside of the catalytic domain of CENP-E may also play a role in regulating the processive behavior of this motor. We have previously shown that the mitotic motor centrosome protein E (CENP-E) is capable of walking for more than 250 steps on its microtubule track without dissociating. We have examined the kinetics of this molecular motor to see if its enzymology explains this remarkable degree of processivity. We find that like the highly processive transport motor kinesin 1, the enzymatic cycle of CENP-E is characterized by rapid ATP binding, multiple enzymatic turnovers per diffusive encounter, and gating of nucleotide binding. These features endow CENP-E with a high duty cycle, a prerequisite for processivity. However, unlike kinesin 1, neck linker docking in CENP-E is slow, occurring at a rate closer to that for Eg5, a mitotic kinesin that takes only 5–10 steps per processive run. These results suggest that like kinesin 1, features outside of the catalytic domain of CENP-E may also play a role in regulating the processive behavior of this motor. IntroductionThe mitotic kinesins are a group of microtubule-based molecular motors that help orchestrate the complex dynamics of chromosomal and spindle movements during the process of mitosis (1.Goshima G. Vale R.D. J. Cell Biol. 2003; 162: 1003-1016Crossref PubMed Scopus (316) Google Scholar). Among these is centrosome protein E (CENP-E). 2The abbreviations used are: CENP-Ecentrosome protein EmantN-methylanthraniloyl2′dmD2′-deoxy-N-methylanthraniloyl-ADP2′dmT2′-deoxy-N-methylanthraniloyl-ATPFlAsH4′,5′-bis(arsenoso)fluoresceinReAsH4,5-bis(1,3,2-dithiarsolan-2-yl)-resorufinAMPPNP5′-adenylyl-β,γ-imidodiphosphateFRETfluorescence resonance energy transferMTmicrotubule. CENP-E is markedly up-regulated during the G2 and M phases of the cell cyle, and it appears to be necessary both for proper chromosomal congression to the metaphase plate and for binding of spindle microtubules to the kinetochore (2.Schaar B.T. Chan G.K. Maddox P. Salmon E.D. Yen T.J. J. Cell Biol. 1997; 139: 1373-1382Crossref PubMed Scopus (263) Google Scholar, 3.Wood K.W. Sakowicz R. Goldstein L.S. Cleveland D.W. Cell. 1997; 91: 357-366Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 4.Yao X. Abrieu A. Zheng Y. Sullivan K.F. Cleveland D.W. Nat. Cell Biol. 2000; 2: 484-491Crossref PubMed Scopus (304) Google Scholar, 5.Putkey F.R. Cramer T. Morphew M.K. Silk A.D. Johnson R.S. McIntosh J.R. Cleveland D.W. Dev. Cell. 2002; 3: 351-365Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Furthermore, it had been proposed that CENP-E functions by actively transporting chromosomes along kinetochore fibers (5.Putkey F.R. Cramer T. Morphew M.K. Silk A.D. Johnson R.S. McIntosh J.R. Cleveland D.W. Dev. Cell. 2002; 3: 351-365Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 6.Weaver B.A. Bonday Z.Q. Putkey F.R. Kops G.J. Silk A.D. Cleveland D.W. J. Cell Biol. 2003; 162: 551-563Crossref PubMed Scopus (213) Google Scholar, 7.McEwen B.F. Chan G.K. Zubrowski B. Savoian M.S. Sauer M.T. Yen T.J. Mol. Biol. Cell. 2001; 12: 2776-2789Crossref PubMed Scopus (207) Google Scholar). This putative role would therefore predict that CENP-E works as a processive transport molecular motor. As such, it would therefore be expected to share features with another, better characterized processive kinesin, kinesin 1. Consistent with this, we had previously shown that a dimeric construct of CENP-E is capable of walking processively for over 250 steps on the microtubule in a hand over hand manner, with stall forces very similar to what had been previously measured for kinesin 1 (8.Yardimci H. van Duffelen M. Mao Y. Rosenfeld S.S. Selvin P.R. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 6016-6021Crossref PubMed Scopus (55) Google Scholar).In the case of kinesin 1, processivity is in part a result of specific features of this motor's enzymology. These include a high duty ratio, rapid ATP binding, the ability to undergo multiple enzymatic turnovers per diffusive encounter, and strain-dependent gating of the motor ATPase (9.Rosenfeld S.S. Fordyce P.M. Jefferson G.M. King P.H. Block S.M. J. Biol. Chem. 2003; 278: 18550-185566Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 10.Hall M.C. Shcherbakova P.V. Kunkel T.A. J. Biol. Chem. 2002; 277: 3673-3679Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 11.Klumpp L.M. Hoenger A. Gilbert S.P. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 3444-3449Crossref PubMed Scopus (96) Google Scholar, 12.Jiang W. Stock M.F. Li X. Hackney D.D. J. Biol. Chem. 1997; 272: 7626-7632Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 13.Jiang W. Hackney D.D. J. Biol. Chem. 1997; 272: 5616-5621Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). These features reduce the probability that the kinesin 1 motor will detach prematurely from its microtubule track. This connection between enzymology and processivity also applies to Eg5, a mitotic kinesin responsible for formation of a bipolar spindle. We had previously shown that neck linker docking for Eg5 gates ATP binding, and it is 20-fold slower than for kinesin 1, and these differences explain the much shorter run lengths for this processive mitotic motor (14.Rosenfeld S.S. Xing J. Jefferson G.M. King P.H. J. Biol. Chem. 2005; 280: 35684-35695Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In this study, we examine the ATPase cycle of CENP-E to see if, like kinesin 1, its enzymology is consistent with its remarkable degree of processivity. IntroductionThe mitotic kinesins are a group of microtubule-based molecular motors that help orchestrate the complex dynamics of chromosomal and spindle movements during the process of mitosis (1.Goshima G. Vale R.D. J. Cell Biol. 2003; 162: 1003-1016Crossref PubMed Scopus (316) Google Scholar). Among these is centrosome protein E (CENP-E). 2The abbreviations used are: CENP-Ecentrosome protein EmantN-methylanthraniloyl2′dmD2′-deoxy-N-methylanthraniloyl-ADP2′dmT2′-deoxy-N-methylanthraniloyl-ATPFlAsH4′,5′-bis(arsenoso)fluoresceinReAsH4,5-bis(1,3,2-dithiarsolan-2-yl)-resorufinAMPPNP5′-adenylyl-β,γ-imidodiphosphateFRETfluorescence resonance energy transferMTmicrotubule. CENP-E is markedly up-regulated during the G2 and M phases of the cell cyle, and it appears to be necessary both for proper chromosomal congression to the metaphase plate and for binding of spindle microtubules to the kinetochore (2.Schaar B.T. Chan G.K. Maddox P. Salmon E.D. Yen T.J. J. Cell Biol. 1997; 139: 1373-1382Crossref PubMed Scopus (263) Google Scholar, 3.Wood K.W. Sakowicz R. Goldstein L.S. Cleveland D.W. Cell. 1997; 91: 357-366Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 4.Yao X. Abrieu A. Zheng Y. Sullivan K.F. Cleveland D.W. Nat. Cell Biol. 2000; 2: 484-491Crossref PubMed Scopus (304) Google Scholar, 5.Putkey F.R. Cramer T. Morphew M.K. Silk A.D. Johnson R.S. McIntosh J.R. Cleveland D.W. Dev. Cell. 2002; 3: 351-365Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Furthermore, it had been proposed that CENP-E functions by actively transporting chromosomes along kinetochore fibers (5.Putkey F.R. Cramer T. Morphew M.K. Silk A.D. Johnson R.S. McIntosh J.R. Cleveland D.W. Dev. Cell. 2002; 3: 351-365Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 6.Weaver B.A. Bonday Z.Q. Putkey F.R. Kops G.J. Silk A.D. Cleveland D.W. J. Cell Biol. 2003; 162: 551-563Crossref PubMed Scopus (213) Google Scholar, 7.McEwen B.F. Chan G.K. Zubrowski B. Savoian M.S. Sauer M.T. Yen T.J. Mol. Biol. Cell. 2001; 12: 2776-2789Crossref PubMed Scopus (207) Google Scholar). This putative role would therefore predict that CENP-E works as a processive transport molecular motor. As such, it would therefore be expected to share features with another, better characterized processive kinesin, kinesin 1. Consistent with this, we had previously shown that a dimeric construct of CENP-E is capable of walking processively for over 250 steps on the microtubule in a hand over hand manner, with stall forces very similar to what had been previously measured for kinesin 1 (8.Yardimci H. van Duffelen M. Mao Y. Rosenfeld S.S. Selvin P.R. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 6016-6021Crossref PubMed Scopus (55) Google Scholar).In the case of kinesin 1, processivity is in part a result of specific features of this motor's enzymology. These include a high duty ratio, rapid ATP binding, the ability to undergo multiple enzymatic turnovers per diffusive encounter, and strain-dependent gating of the motor ATPase (9.Rosenfeld S.S. Fordyce P.M. Jefferson G.M. King P.H. Block S.M. J. Biol. Chem. 2003; 278: 18550-185566Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 10.Hall M.C. Shcherbakova P.V. Kunkel T.A. J. Biol. Chem. 2002; 277: 3673-3679Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 11.Klumpp L.M. Hoenger A. Gilbert S.P. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 3444-3449Crossref PubMed Scopus (96) Google Scholar, 12.Jiang W. Stock M.F. Li X. Hackney D.D. J. Biol. Chem. 1997; 272: 7626-7632Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 13.Jiang W. Hackney D.D. J. Biol. Chem. 1997; 272: 5616-5621Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). These features reduce the probability that the kinesin 1 motor will detach prematurely from its microtubule track. This connection between enzymology and processivity also applies to Eg5, a mitotic kinesin responsible for formation of a bipolar spindle. We had previously shown that neck linker docking for Eg5 gates ATP binding, and it is 20-fold slower than for kinesin 1, and these differences explain the much shorter run lengths for this processive mitotic motor (14.Rosenfeld S.S. Xing J. Jefferson G.M. King P.H. J. Biol. Chem. 2005; 280: 35684-35695Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In this study, we examine the ATPase cycle of CENP-E to see if, like kinesin 1, its enzymology is consistent with its remarkable degree of processivity.