Regulation of Microtubule Destabilizing Activity of Op18/Stathmin Downstream of Rac1

Abstract

In the leading edge of migrating cells, a subset of microtubules exhibits net growth in a Rac1- and p21-activated kinase-dependent manner. Here, we explore the possibility of whether phosphorylation and inactivation of the microtubule-destabilizing protein Op18/stathmin could be a mechanism regulating microtubule dynamics downstream of Rac1 and p21-activated kinases. We find that, in vitro, Pak1 phosphorylates Op18/stathmin specifically at serine 16 and inactivates its catastrophe promoting activity in biochemical and time lapse microscopy microtubule assembly assays. Furthermore, phosphorylation of either serine 16 or 63 is sufficient to inhibit Op18/stathmin in vitro. In cells, the microtubule-destabilizing effect of an excess of Op18/stathmin can be partially overcome by expression of constitutively active Rac1(Q61L), which is dependent on Pak activity, suggesting that the microtubule cytoskeleton can be regulated through inactivation of Op18/stathmin downstream of Rac1 and Pak in vivo. However, in vivo, Pak1 activity alone is not sufficient to phosphorylate Op18, indicating that additional pathways downstream of Rac1 are required for Op18 regulation. In the leading edge of migrating cells, a subset of microtubules exhibits net growth in a Rac1- and p21-activated kinase-dependent manner. Here, we explore the possibility of whether phosphorylation and inactivation of the microtubule-destabilizing protein Op18/stathmin could be a mechanism regulating microtubule dynamics downstream of Rac1 and p21-activated kinases. We find that, in vitro, Pak1 phosphorylates Op18/stathmin specifically at serine 16 and inactivates its catastrophe promoting activity in biochemical and time lapse microscopy microtubule assembly assays. Furthermore, phosphorylation of either serine 16 or 63 is sufficient to inhibit Op18/stathmin in vitro. In cells, the microtubule-destabilizing effect of an excess of Op18/stathmin can be partially overcome by expression of constitutively active Rac1(Q61L), which is dependent on Pak activity, suggesting that the microtubule cytoskeleton can be regulated through inactivation of Op18/stathmin downstream of Rac1 and Pak in vivo. However, in vivo, Pak1 activity alone is not sufficient to phosphorylate Op18, indicating that additional pathways downstream of Rac1 are required for Op18 regulation. Tight regulation of cytoskeletal dynamics in living cells is essential for many cellular behaviors such as mitosis or motility. Rho family GTPases are well characterized regulators of actin cytoskeleton reorganization in response to extracellular signals (1Ridley A.J. J. Cell Sci. 2001; 114: 2713-2722Crossref PubMed Google Scholar). However, recent evidence has also linked Rho GTPases to regulation of the dynamics of the microtubule cytoskeleton (2Wittmann T. Waterman-Storer C.M. J. Cell Sci. 2001; 114: 3795-3803Crossref PubMed Google Scholar). For example, RhoA and its downstream effector, mDia, have been implicated in the formation of stable, detyrosinated microtubules (3Cook T.A. Nagasaki T. Gundersen G.G. J. Cell Biol. 1998; 141: 175-185Crossref PubMed Scopus (211) Google Scholar, 4Palazzo A.F. Cook T.A. Alberts A.S. Gundersen G.G. Nat. Cell Biol. 2001; 3: 723-729Crossref PubMed Scopus (476) Google Scholar), whereas Cdc42, together with cytoplasmic dynein, is involved in the orientation of the microtubule-organizing center toward the leading edge of migrating cells (5Palazzo A.F. Joseph H.L. Chen Y.J. Dujardin D.L. Alberts A.S. Pfister K.K. Vallee R.B. Gundersen G.G. Curr. Biol. 2001; 11: 1536-1541Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 6Etienne-Manneville S. Hall A. Cell. 2001; 106: 489-498Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar). In addition, activation of Rac1 promotes microtubule growth into advancing lamellipodia of migrating cells (7Wittmann T. Bokoch G.M. Waterman-Storer C.M. J. Cell Biol. 2003; 161: 845-851Crossref PubMed Scopus (216) Google Scholar). In cells and in vitro, microtubules exhibit a nonequilibrium polymerization behavior known as dynamic instability, the stochastic switching between phases of growth and shortening (8Desai A. Mitchison T.J. Annu. Rev. Cell Dev. Biol. 1997; 13: 83-117Crossref PubMed Scopus (1937) Google Scholar). Dynamic instability can be described by four parameters: the rates of growth and shortening and the transition frequencies from growth to shortening (catastrophe frequency) and from shortening to growth (rescue frequency). In vivo, dynamic instability is regulated by microtubule-associated proteins, microtubule plus end-binding proteins, and soluble proteins, creating a balance between microtubule stabilizing and destabilizing factors that promote the rapid turnover of microtubules required for cellular morphogenesis (9Andersen S.S. Wittmann T. Bioessays. 2002; 24: 305-307Crossref PubMed Scopus (15) Google Scholar, 10Kinoshita K. Habermann B. Hyman A.A. Trends Cell Biol. 2002; 12: 267-273Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 11Rodriguez O.C. Schaefer A.W. Mandato C.A. Forscher P. Bement W.M. Waterman-Storer C.M. Nat. Cell Biol. 2003; 5: 599-609Crossref PubMed Scopus (705) Google Scholar). One soluble microtubule-destabilizing protein is Op18/stathmin (Op18) 1The abbreviations used are: Op18Op18/stathminEGFepidermal growth factorPakp21-activated kinasePKAcAMP-dependent kinaseGSTglutathione S-transferasePipes1,4-piperazinediethanesulfonic acid.1The abbreviations used are: Op18Op18/stathminEGFepidermal growth factorPakp21-activated kinasePKAcAMP-dependent kinaseGSTglutathione S-transferasePipes1,4-piperazinediethanesulfonic acid. (12Cassimeris L. Curr. Opin. Cell Biol. 2002; 14: 18-24Crossref PubMed Scopus (363) Google Scholar, 13Andersen S.S. Trends Cell Biol. 2000; 10: 261-267Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Op18 binds tubulin dimers, inhibits tubulin polymerization, promotes catastrophes, and modulates tubulin GTP hydrolysis (14Belmont L.D. Mitchison T.J. Cell. 1996; 84: 623-631Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar, 15Jourdain L. Curmi P. Sobel A. Pantaloni D. Carlier M.F. Biochemistry. 1997; 36: 10817-10821Crossref PubMed Scopus (213) Google Scholar, 16Howell B. Larsson N. Gullberg M. Cassimeris L. Mol. Biol. Cell. 1999; 10: 105-118Crossref PubMed Scopus (158) Google Scholar, 17Segerman B. Holmfeldt P. Morabito J. Cassimeris L. Gullberg M. J. Cell Sci. 2003; 116: 197-205Crossref PubMed Scopus (16) Google Scholar, 18Larsson N. Segerman B. Howell B. Fridell K. Cassimeris L. Gullberg M. J. Cell Biol. 1999; 146: 1289-1302Crossref PubMed Scopus (60) Google Scholar). Although the mechanism by which Op18 induces microtubule catastrophes is not entirely understood, it appears that this activity is at least partially separable from its ability to sequester tubulin dimer (16Howell B. Larsson N. Gullberg M. Cassimeris L. Mol. Biol. Cell. 1999; 10: 105-118Crossref PubMed Scopus (158) Google Scholar, 17Segerman B. Holmfeldt P. Morabito J. Cassimeris L. Gullberg M. J. Cell Sci. 2003; 116: 197-205Crossref PubMed Scopus (16) Google Scholar). Mammalian Op18 is regulated by phosphorylation at four serine residues by a number of protein kinases, and dual phosphorylation particularly of serines 16 and 63 inhibits the microtubule destabilizing activity of Op18 (19Larsson N. Marklund U. Gradin H.M. Brattsand G. Gullberg M. Mol. Cell. Biol. 1997; 17: 5530-5539Crossref PubMed Scopus (168) Google Scholar, 20Melander Gradin H. Marklund U. Larsson N. Chatila T.A. Gullberg M. Mol. Cell. Biol. 1997; 17: 3459-3467Crossref PubMed Scopus (124) Google Scholar, 21Melander Gradin H. Larsson N. Marklund U. Gullberg M. J. Cell Biol. 1998; 140: 131-141Crossref PubMed Scopus (122) Google Scholar, 22Horwitz S.B. Shen H.J. He L. Dittmar P. Neef R. Chen J. Schubart U.K. J. Biol. Chem. 1997; 272: 8129-8132Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Op18/stathmin epidermal growth factor p21-activated kinase cAMP-dependent kinase glutathione S-transferase 1,4-piperazinediethanesulfonic acid. Op18/stathmin epidermal growth factor p21-activated kinase cAMP-dependent kinase glutathione S-transferase 1,4-piperazinediethanesulfonic acid. Op18 becomes phosphorylated in various cancer cell lines in response to treatment with epidermal growth factor (EGF) (23Daub H. Gevaert K. Vandekerckhove J. Sobel A. Hall A. J. Biol. Chem. 2001; 276: 1677-1680Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 24Hoelscher S.R. Ascoli M. Endocrinology. 1994; 135: 662-666Crossref PubMed Scopus (5) Google Scholar, 25Ji H. Baldwin G.S. Burgess A.W. Moritz R.L. Ward L.D. Simpson R.J. J. Biol. Chem. 1993; 268: 13396-13405Abstract Full Text PDF PubMed Google Scholar), and it has recently been shown that EGF-stimulated phosphorylation at serine 16 requires the activity of Rac1 and p21-activated serine/threonine kinases (Paks) (23Daub H. Gevaert K. Vandekerckhove J. Sobel A. Hall A. J. Biol. Chem. 2001; 276: 1677-1680Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 26Bokoch G.M. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (877) Google Scholar). However, the physiological significance of this is unknown. In migrating cells, a subset of microtubules exhibits increased growth into the advancing cell edge of a catastrophe and an increased time microtubules in the growth (7Wittmann T. Bokoch G.M. Waterman-Storer C.M. J. Cell Biol. 2003; 161: 845-851Crossref PubMed Scopus (216) Google Scholar, C.M. J. Cell Biol. 1997; PubMed Scopus Google Scholar). we have that expression of constitutively active in microtubule net growth in cellular microtubules and that this on Pak kinase activity (7Wittmann T. Bokoch G.M. Waterman-Storer C.M. J. Cell Biol. 2003; 161: 845-851Crossref PubMed Scopus (216) Google Scholar). One possibility is that this of microtubule growth could be by Pak kinase regulation of the microtubule destabilizing activity of We find that Pak1 can phosphorylate Op18 at serine 16 and inhibit its catastrophe promoting activity in vitro, in Pak kinase activity alone is not sufficient to phosphorylate In addition, of downstream of microtubule destabilizing activity of Op18 in cells, indicating that Op18 activity can be regulated by in vivo. and expression or a from and and by glutathione as described S. H.J. J. Bokoch G.M. PubMed Scopus Google Scholar, Bokoch G.M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). and Op18, of Op18 and proteins, and of Gullberg of for phosphorylated serine 16 from Sobel Op18 by with and at the and and into the J. M. M. L. Biochem. 1996; PubMed Scopus Google Scholar). protein the to the of Op18 in cells and on to the In and or phosphorylated with in and or with in and at at for and by on or and For in microtubule polymerization Op18 phosphorylated in with kinase and the for cells with Op18 phosphorylation on Op18 from the cell by of cellular and of the Op18 from the with and at in a of In kinase with Pak1 as described Bokoch G.M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). kinase from the effect of Op18 on microtubule polymerization, tubulin M.F. J. Cell Biol. PubMed Scopus Google with Op18 in of and microtubules at for and from tubulin dimer by at for at of and to with and on a In Cell Biol. 24: PubMed Scopus Google in into a of a with of on a on and to with of tubulin and GTP in the and to the that been to with an on an with a a and a in with an and such that a of could be in such and for microtubule and dynamic instability by microtubule as the microtubules from the M.F. J. Cell Biol. PubMed Scopus Google Scholar). of to in cells and as described (7Wittmann T. Bokoch G.M. Waterman-Storer C.M. J. Cell Biol. 2003; 161: 845-851Crossref PubMed Scopus (216) Google Scholar). microtubule as described G.G. J. Cell Sci. 1994; PubMed Google Scholar). on with tubulin at cells with a with the and tubulin dimers, the cells with and in the to inhibit and the the at tubulin in cells and and and the of tubulin to soluble dimer of the with and for Pak1 Op18 at Op18 phosphorylation downstream of is dependent on Pak kinase activity in cells (23Daub H. Gevaert K. Vandekerckhove J. Sobel A. Hall A. J. Biol. Chem. 2001; 276: 1677-1680Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar), we to whether Pak could phosphorylate Op18 in vitro. For this Op18 with constitutively active S. H.J. J. Bokoch G.M. PubMed Scopus Google in the of Although in phosphorylation by of the activity of the the of Op18 phosphorylation with the of suggesting that Op18 is a physiological of of or used as a that Pak1 phosphorylates Op18 at serine the of Op18 phosphorylation by we in phosphorylated and Op18 by Op18 and phosphorylated by at a However, not that Pak1 is for serine phosphorylation on serine 63 a to a phosphorylation with Op18 or as a with a phosphorylated Op18 at serines 16 and as described Gradin H. Larsson N. Marklund U. Gullberg M. J. Cell Biol. 1998; 140: 131-141Crossref PubMed Scopus (122) Google and a for serine 63 serine we also that Pak1 and phosphorylated the of Op18 not suggesting that phosphorylation of family and by Pak1 Op18 in whether phosphorylation by Pak1 the ability of Op18 to inhibit the polymerization of tubulin in (19Larsson N. Marklund U. Gradin H.M. Brattsand G. Gullberg M. Mol. Cell. Biol. 1997; 17: 5530-5539Crossref PubMed Scopus (168) Google Scholar). dimer with of Op18, and microtubule polymerization by the of GTP and and at microtubules from tubulin dimer by Op18 tubulin polymerization, whereas Op18 that been phosphorylated with Pak1 microtubule polymerization at Op18 the of tubulin in the a phosphorylation with Op18 Gradin H. Larsson N. Marklund U. Gullberg M. J. Cell Biol. 1998; 140: 131-141Crossref PubMed Scopus (122) Google Scholar). whether inactivation of the microtubule destabilizing activity of Op18 to phosphorylation at serine Op18 and phosphorylated with Pak1 for of and on tubulin polymerization of Op18 with the of the phosphorylation whereas of with Pak1 for effect on its ability to inhibit microtubule polymerization that Pak1 phosphorylation at serine 16 is and sufficient to inhibit the microtubule destabilizing activity of Op18 in vitro. such a tubulin polymerization not between tubulin dimer and catastrophe we also the effect of Pak1 phosphorylation of Op18 on microtubule plus dynamic instability microtubules from at tubulin and by at a at which of Op18 to tubulin is S.S. S. M. Sobel A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, P. Larsson N. Segerman B. Howell B. Morabito J. Cassimeris L. Gullberg M. Mol. Biol. Cell. 2001; 12: PubMed Scopus Google Scholar, P. Pantaloni D. Carlier M.F. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), and Op18 the plus catastrophe of its on dimer and growth (16Howell B. Larsson N. Gullberg M. Cassimeris L. Mol. Biol. Cell. 1999; 10: 105-118Crossref PubMed Scopus (158) Google Scholar). the of or Op18 increased the catastrophe or and We also a in plus growth that at Op18 by of In as as Op18 effect on either plus catastrophe or growth and that phosphorylation by Pak1 alone can the catastrophe promoting activity of by Pak1 inactivates of of Op18, Op18, in a Op18 phosphorylation microtubule dynamics in has been with protein that been dual phosphorylated on serines 16 and 63 P. Larsson N. Segerman B. Howell B. Morabito J. Cassimeris L. Gullberg M. Mol. Biol. Cell. 2001; 12: PubMed Scopus Google Scholar). Pak1 phosphorylated Op18 at serine we to the of phosphorylation at either serine 16 or 63 to Op18 For this we plus microtubule dynamic instability in in the of either or phosphorylation increased the catastrophe with phosphorylation of with in phosphorylation of either serine 63 or the catastrophe promoting activity of that phosphorylation at either serine 16 or 63 is sufficient to Op18 in and of either serine 16 or serine 63 inactivates of of in a of Op18 of Rac1 in of Op18 in response to has been in cancer this microtubules in cells has not been (23Daub H. Gevaert K. Vandekerckhove J. Sobel A. Hall A. J. Biol. Chem. 2001; 276: 1677-1680Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 24Hoelscher S.R. Ascoli M. Endocrinology. 1994; 135: 662-666Crossref PubMed Scopus (5) Google Scholar, 25Ji H. Baldwin G.S. Burgess A.W. Moritz R.L. Ward L.D. Simpson R.J. J. Biol. Chem. 1993; 268: 13396-13405Abstract Full Text PDF PubMed Google Scholar). whether such growth Op18 phosphorylation could be for the regulation of microtubule in vivo, we the of Op18 phosphorylation in cells, the cell in which we have increased microtubule growth downstream of Rac1 activation (7Wittmann T. Bokoch G.M. Waterman-Storer C.M. J. Cell Biol. 2003; 161: 845-851Crossref PubMed Scopus (216) Google Scholar). cells that been with for and by with an or an for phosphorylated serine 16 S. S. Curmi P. Sobel A. J. Cell Sci. 1998; PubMed Google Scholar). We serine 16 phosphorylation and phosphorylation However, the of Op18 that phosphorylated as by the of of that and by the In addition, that treatment induces phosphorylation at serine 16 as shown (23Daub H. Gevaert K. Vandekerckhove J. Sobel A. Hall A. J. Biol. Chem. 2001; 276: 1677-1680Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). We that expression of constitutively active of Pak1 not sufficient to the net microtubule growth in cells (7Wittmann T. Bokoch G.M. Waterman-Storer C.M. J. Cell Biol. 2003; 161: 845-851Crossref PubMed Scopus (216) Google Scholar). we to whether constitutively active Pak1 can phosphorylate Op18 in vivo. For this we used cells, could be and phosphorylation of Op18 at serine 16 in cells either constitutively active Rac1(Q61L), or constitutively active Pak1 or J. J. Cell Biol. 1999; PubMed Scopus Google Scholar). that Op18 phosphorylation at serine 16 increased in cells not in cells either of the constitutively active Pak1 whether the Pak1 active in cells, we Pak1 from cells and its activity in a kinase that active and of Op18 in whether kinases that are known to phosphorylate Op18 at serine 16 are we used kinase and effect on Op18 phosphorylation in cells and Op18 phosphorylation in cells not H. M. P. Biochem. J. 2000; PubMed Scopus Google Scholar). a of kinases, a of and Op18 indicating that kinases not phosphorylate Op18 downstream of Furthermore, to effect on microtubule growth in cells not (7Wittmann T. Bokoch G.M. Waterman-Storer C.M. J. Cell Biol. 2003; 161: 845-851Crossref PubMed Scopus (216) Google Scholar). In addition, a of phosphorylation at serine 16 suggesting that is not a of serine 16 phosphorylation in of protein kinase and of which have been implicated in the of the microtubule cytoskeleton in migrating cells A. K. T. B. T. N. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Hall A. 2003; PubMed Scopus Google Scholar), also of Op18 that Pak activity is required for Op18 phosphorylation (23Daub H. Gevaert K. Vandekerckhove J. Sobel A. Hall A. J. Biol. Chem. 2001; 276: 1677-1680Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar), it is not sufficient on its However, kinases known to phosphorylate serine 16 are not of Rac1 Op18 in serine 16 phosphorylation of Op18 downstream of Rac1 on Pak1 activity (23Daub H. Gevaert K. Vandekerckhove J. Sobel A. Hall A. J. Biol. Chem. 2001; 276: 1677-1680Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar), we to whether the of Rac1 activation on the microtubule cytoskeleton could be linked to inactivation of Op18 in we that activation of Rac1 in inactivation of Op18 and promote an in the of tubulin in cells B. H. Cassimeris L. J. Cell Sci. 1999; Google Scholar). we the of microtubule to tubulin dimer in cells G.G. J. Cell Sci. 1994; PubMed Google Scholar). living cells with tubulin and to the tubulin in the of soluble and with a and a to the of tubulin into microtubules In cells, of the tubulin into microtubule to has been described for cells G.G. J. Cell Sci. 1994; PubMed Google Scholar). of effect whereas expression of constitutively active the to dimer microtubule and turnover in cells (7Wittmann T. Bokoch G.M. Waterman-Storer C.M. J. Cell Biol. 2003; 161: 845-851Crossref PubMed Scopus (216) Google Scholar). Although Op18 is its are in many from that cells of of soluble protein to which is with cell B. H. Cassimeris L. J. Cell Sci. 1999; Google Scholar, G. G. Marklund U. H. G. P. Gullberg M. 1993; Google Scholar). inactivation of Op18 not be to a effect on the of microtubule in whether Rac1 of regulating the microtubule cytoskeleton through Op18 in vivo, we increased the Op18 by to an of an of of the of the which is to the cellular tubulin of such excess Op18 the of tubulin in and in active cells However, in cells with the of Op18, the of tubulin to indicating that Op18 is downstream of Furthermore, we active cells with phosphorylation the microtubules to a as in cells with Op18 and that Rac1 can the microtubule destabilizing activity of Op18 in and that this on serine on in that Pak1 phosphorylated and Op18, we to whether Pak activity required downstream of Rac1 to Op18 in vivo. For this we and active cells tubulin with a of that inhibits the kinase activity of in sequester Rac1 Bokoch G.M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). alone in with active the microtubule as with cells However, we cells with a of Op18 and Pak the microtubule in cells to 16 and to in active cells which is we in cells with Op18 alone and to the in cells with and Op18 or active and that the microtubule destabilizing activity of Op18 can be in downstream of Rac1 by phosphorylation at serine In this we have the possibility of whether inactivation of the microtubule destabilizing protein Op18 through phosphorylation downstream of Rac1 and could be for the Rac1- and regulation of leading edge microtubule dynamics that we have in (7Wittmann T. Bokoch G.M. Waterman-Storer C.M. J. Cell Biol. 2003; 161: 845-851Crossref PubMed Scopus (216) Google Scholar). We find that Op18 is specifically phosphorylated in by Pak1 at serine of Pak1 in is with the of (23Daub H. Gevaert K. Vandekerckhove J. Sobel A. Hall A. J. Biol. Chem. 2001; 276: 1677-1680Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar), Rac1- and phosphorylation at serine 16 in EGF-stimulated by Pak1 is sufficient to the activity of Op18 on tubulin polymerization as well as its plus catastrophe promoting activity as by dynamic instability of microtubules in by from in which of Op18 phosphorylation on microtubule polymerization have been with Op18 protein that been phosphorylated on (19Larsson N. Marklund U. Gradin H.M. Brattsand G. Gullberg M. Mol. Cell. Biol. 1997; 17: 5530-5539Crossref PubMed Scopus (168) Google Scholar, 21Melander Gradin H. Larsson N. Marklund U. Gullberg M. J. Cell Biol. 1998; 140: 131-141Crossref PubMed Scopus (122) Google Scholar, P. Larsson N. Segerman B. Howell B. Morabito J. Cassimeris L. Gullberg M. Mol. Biol. Cell. 2001; 12: PubMed Scopus Google Scholar). phosphorylation of serines 16 and 63 by inhibits the catastrophe promoting activity of Op18 in P. Larsson N. Segerman B. Howell B. Morabito J. Cassimeris L. Gullberg M. Mol. Biol. Cell. 2001; 12: PubMed Scopus Google Scholar). Op18 a of phosphorylation in (12Cassimeris L. Curr. Opin. Cell Biol. 2002; 14: 18-24Crossref PubMed Scopus (363) Google Scholar), it has been that phosphorylation is required to We find that, in vitro, of either serine 16 or 63 is sufficient to inhibit the catastrophe promoting activity of is with the that of protein which is also for serine inhibits the microtubule destabilizing activity of Op18 in cells Gradin H. Marklund U. Larsson N. Chatila T.A. Gullberg M. Mol. Cell. Biol. 1997; 17: 3459-3467Crossref PubMed Scopus (124) Google Scholar, S. Sobel A. J. 1998; 161: Google and that serine 63 phosphorylation of a Op18 its H. H. D. J. 2001; PubMed Scopus Google Scholar). 16 is the phosphorylation through of the protein family (12Cassimeris L. Curr. Opin. Cell Biol. 2002; 14: 18-24Crossref PubMed Scopus (363) Google Scholar). However, we that the least of proteins, is not phosphorylated by Pak1 or suggesting that are between In vivo, we Op18 phosphorylation at serine 16 downstream of treatment or expression of constitutively active active not phosphorylate Op18 in vivo, the kinase active in vitro. that in vivo, additional factors downstream of Rac1 are required for Op18 is with that constitutively active Pak1 not have the effect on microtubule growth as constitutively active Rac1 (7Wittmann T. Bokoch G.M. Waterman-Storer C.M. J. Cell Biol. 2003; 161: 845-851Crossref PubMed Scopus (216) Google and of that Pak activity alone is not sufficient to cytoskeletal A. S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, L. D. R. Nat. Cell Biol. 2002; PubMed Scopus Google Scholar). We not additional factors are required for Op18 that and protein which can phosphorylate Op18 at serine are not One possibility is that of Pak to the cells leading edge is required A. J. J. Cell Biol. 2000; PubMed Scopus Google Scholar), which we not with constitutively active of Wittmann and M. of cellular microtubule that Rac1 and of are of regulating Op18 activity in vivo. However, we to Rac1- or on microtubule in the of excess such Op18 to the cellular tubulin tubulin dimer is to be catastrophe whereas at physiological Op18 the catastrophe be regulated through Op18 of cellular microtubule is not to such In addition, Rac1 activation also microtubule turnover and by increased and microtubule the of microtubule (7Wittmann T. Bokoch G.M. Waterman-Storer C.M. J. Cell Biol. 2003; 161: 845-851Crossref PubMed Scopus (216) Google Scholar, Waterman-Storer C.M. Curr. Biol. 2002; 12: Full Text Full Text PDF PubMed Scopus Google Scholar). also the of microtubule in the of excess Op18 increased in cells as with of Op18 regulation is also with the of Op18 that becomes phosphorylated and H. Gevaert K. Vandekerckhove J. Sobel A. Hall A. J. Biol. Chem. 2001; 276: 1677-1680Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar), which not be to have a on microtubule In we not to an effect of on microtubule dynamics in Wittmann and M. a to in living cells has been used to that Op18 is to tubulin in the leading edge of P. and in a migrating a subset of to as exhibits a catastrophe in increased net growth into the edge of the cell (7Wittmann T. Bokoch G.M. Waterman-Storer C.M. J. Cell Biol. 2003; 161: 845-851Crossref PubMed Scopus (216) Google Scholar, C.M. J. Cell Biol. 1997; PubMed Scopus Google Scholar). that regulation of a of Op18 by active Rac1 and Pak at the leading edge could to microtubule behavior A. J. J. Cell Biol. 2000; PubMed Scopus Google Scholar, Bokoch G.M. S. 2000; PubMed Scopus Google Scholar). microtubule behavior is in microtubules in cells constitutively active (7Wittmann T. Bokoch G.M. Waterman-Storer C.M. J. Cell Biol. 2003; 161: 845-851Crossref PubMed Scopus (216) Google with a inactivation of a microtubule polymerization such as However, downstream of Rac1 Pak are involved in the regulation of leading edge microtubule Pak activity is not sufficient for of microtubule growth (7Wittmann T. Bokoch G.M. Waterman-Storer C.M. J. Cell Biol. 2003; 161: 845-851Crossref PubMed Scopus (216) Google Scholar). Dynamic between the microtubule and actin cytoskeleton be required for microtubule behavior to O.C. Schaefer A.W. Mandato C.A. Forscher P. Bement W.M. Waterman-Storer C.M. Nat. Cell Biol. 2003; 5: 599-609Crossref PubMed Scopus (705) Google Scholar). possibility is that Rac1 and inactivation of Op18 microtubule growth by the catastrophe which could be by microtubule through of plus end-binding such as or A. K. T. B. T. N. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, M. T. J. M. M. S. A. K. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, A.S. S. N. G.G. J. Cell Biol. 2002; PubMed Scopus Google Scholar, S. Mitchison T.J. Mol. Biol. Cell. 2002; 13: PubMed Scopus Google Scholar, J. K. Hyman A.A. Curr. Biol. 2001; 11: Full Text Full Text PDF PubMed Scopus (363) Google Scholar). of protein kinase and which have been to be involved in the of microtubules through plus end-binding A. K. T. B. T. N. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Hall A. 2003; PubMed Scopus Google effect on Op18 it is that are pathways regulating microtubule in migrating We and Sobel for and of the Waterman-Storer for and on the particularly for the of