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Microtubule dynamics: If you need a shrink try /Op18 Sean Lawler

Recent studies show that stathmin/Op18 may be an kinase A (PKA), and Ser16 by the Ca2+/calmodulin- important physiological regulator of dependent kinase-Gr (CaMK IV/Gr). There are almost dynamics; the activity of stathmin/Op18 is controlled by certainly additional unidentified kinases which phospho- the actions of several signalling pathways, allowing it to rylate stathmin/Op18. play a central role in coordinating microtubule behaviour. Modulation of stathmin/Op18 levels by overexpression or Address: MRC Unit, Department of Biochemistry, Dundee University, DD1 4HN, UK. with anti-sense cDNAs results in a striking -cycle block at the G2/M transition, suggesting an involvement Current Biology 1998, 8:R212–R214 in cell-cycle progression [5]. The function of http://biomednet.com/elecref/09609822008R0212 stathmin/Op18 remained elusive, however, until the © Current Biology Ltd ISSN 0960-9822 protein was purified in a screen for factors that could inhibit microtubule polymerisation [6]. This showed that, The regulation of microtubule dynamics in eukaryotic in vitro, stathmin/Op18 interacts with unpolymerised cells is essential for a multitude of processes, such as cell and destabilises , and conversely that movement, morphology, division and cytoplasmic its depletion from Xenopus egg extracts increases micro- organisation [1]. Microtubules are dynamic of αβ tubule formation. These effects were attributed to an tubulin dimers, the behaviour of which is determined by a increased microtubule catastrophe rate, and may underlie combination of the rates of growth, shrinkage, rescue the previously observed cell-cycle blockade by stath- (change from a shrinking to a growing state) and min/Op18. These observations were a major breakthrough catastrophe (change from a growing to a shrinking state). in stathmin/Op18 research, and rapid progress has since This probably involves the coordinated action of several been made which has shown that stathmin/Op18 is a phos- , but few have been identified and their mode of phorylation-controlled general physiological regulator of action is unclear [2]. The protein stathmin/Op18, microtubule dynamics. however, has recently emerged as a potentially crucial regulator of microtubule dynamics. Phosphorylation and microtubule destabilisation It is now well established that stathmin/Op18 inhibits micro- Stathmin/Op18 — a brief history tubule polymerisation and also depolymerises microtubules Stathmin/Op18 is a 19 kDa cytoplasmic protein that has in a dose-dependent fashion; importantly, this has been been independently isolated by a number of investigators demonstrated in living cells [7,8]. Furthermore, phosphory- in the past few years [3–5]. Various observations indicated lation of stathmin/Op18 on all four sites completely blocks that stathmin/Op18 plays an important role in cell its microtubule-destabilising effect [9]. Thus, unphosphory- regulation. The protein is well conserved and highly lated stathmin/Op18 promotes microtubule disassembly, expressed in proliferating cells and . Its expression and this activity is turned off by phosphorylation. level changes markedly during development, cellular dif- ferentiation and tissue regeneration, and is elevated in Overexpression of mutant forms of stathmin/Op18 in some neoplastic cells. Particularly notable are the diverse which the phosphorylation sites are replaced by alanines phosphoforms of stathmin/Op18 which have been causes a major G2/M block [4] and destabilises micro- observed, the result of phosphorylation on four serine tubules [7,8]. Interestingly, wild-type stathmin/Op18 also residues that is stimulated by a wide range of extracellular destabilises microtubules, but the cells effectors and during . progress apparently normally through the . This difference occurs because the elevated kinase levels that Stathmin/Op18 consists of a carboxy-terminal α-helical occur during mitosis are enough to phosphorylate and domain and an amino-terminal ‘regulatory’ domain that thereby inactivate the overexpressed wild-type protein, contains the four serine phosphorylation sites (residues 16, whereas the alanine mutants cannot be phosphorylated 25, 38 and 63) that account for all the phosphoforms and are constitutively active [7]. Phosphorylation- observed [3]. These sites are the targets of multiple mediated down regulation of stathmin/Op18 activity is kinases regulated both during the cell cycle and by signal thus required for progression through mitosis. transduction cascades. Ser25 is a target for mitogen-acti- vated protein (MAP) kinase, and both Ser25 and Ser38 are Experiments using in vitro phosphorylated stathmin/Op18 targets for phosphorylation by -dependent kinases or mutated forms have shown that its regulation may be (Cdks). Ser16 and Ser63 can be phosphorylated by protein quite complex [9] (see Table 1). It appears that Ser16 and Dispatch R213

Ser63 make the greatest contribution to stathmin/Op18 Table 1 inactivation, a single aspartic acid substitution at Ser63 Stathmin/Op18 phosphorylation effects on microtubule stability. being sufficient to block the microtubule depolymerisa- tion effect in microinjected cells [8]. Phosphorylation of Microtubule both Ser16 and Ser63 has a greater effect on microtubule Sites phosphorylated destabilisation stability in vitro than that of either site alone, suggesting 16 25 38 63 an element of cooperativity. In contrast, phosphorylation of Ser25 and Ser38 has little effect on microtubules in – – – – + + + + vitro. Indeed, overexpression of the Ala16/Ala63 mutant – P P – + + + has a strong destabilising effect on microtubules, despite P––– + + being efficiently phosphorylated at both Ser25 and Ser38 – – – P + + by mitotic kinases [9]. PP P – + –PPP +/– A clue to the function of Ser25 and Ser38 phosphorylation P– – P – comes from overexpression of Ala25/Ala38 mutant stath- PPPP – min/Op18, which gives a strong cell-cycle block, and Stathmin/Op18 was phosphorylated in vitro on the sites marked, and causes microtubule depolymerisation [7]. It turns out that, the effects on microtubule polymerisation were observed in an in vitro in this mutant, Ser16 and Ser63 do not become phospho- assay (from [9]). rylated, probably accounting for its effects, and indicating a necessity for ordered phosphorylation in stathmin/Op18 that stathmin/Op18 causes spindle disassembly in vitro inactivation. Thus, Ser16 and Ser63 regulate microtubule [6,14] and that, in cells expressing alanine mutants of destabilisation, and phosphorylation of Ser25/Ser38 is stathmin/Op18, the spindle does not form or has abnor- required for Ser16/Ser63 phosphorylation, at least during mally short arms [7,9]. The microtubule-destabilising mitosis. All the sites can be phosphorylated efficiently in effect of these mutant proteins thus appears to prevent vitro, so it may be in the tubulin-bound form that sequen- proper spindle formation. Two recent studies using tial phosphorylation is required for stathmin/Op18 inacti- Xenopus egg extracts support a role for stathmin/Op18 in vation, Ser25 and/or Ser38 phosphorylation allowing spindle formation [13,14]. Inhibition of protein phos- access to the other two sites. phatase 2A (PP2A) by okadaic acid was found to cause excessive spindle microtubule growth in extracts, which Stathmin/Op18 interacts directly with αβ tubulin dimers correlated with a decreased catastrophe frequency and [6,9,10,11], and no interaction has been observed between could be rescued by the addition of a constitutively-active stathmin/Op18 and microtubules. A 217 kDa complex stathmin/Op18 mutant [13]. The catastrophe frequency consisting of two tubulin heterodimers per stathmin/Op18 also decreased on immunodepletion of stathmin/Op18 molecule was observed [10,11], and the interaction from the extracts, suggesting that stathmin/Op18 may be a appears to occur primarily between stathmin/Op18 and α- substrate of PP2A in microtubule regulation, mediating at tubulin [9]. The region of stathmin/Op18 necessary for least some of its effects on microtubule stability [13]. microtubule interaction has not been determined, but deletion of the amino-terminal 54 amino acids destroys its Stathmin/Op18 is known to be highly phosphorylated in ability to depolymerise microtubules [7]. mitosis, so it was surprising that its phosphorylation was at a similar low level in both mitotic and interphase Xenopus Whether stathmin/Op18 is truly a microtubule catastrophe egg extracts [14]. However, the addition of increasing factor, as originally proposed [6], is still not clear. Some amounts of mitotic chromatin to the mitotic extract caused studies have found a correlation between stathmin/Op18 a dose-dependent increase in stathmin/Op18 phosphoryla- and catastrophe frequency [12,13], but others have not tion. Mitotic chromatin thus contains an activity that can [10]. Stathmin/Op18 could simply sequester αβ tubulin render a mitotic extract capable of hyperphosphorylating dimers and thereby alter the equilibrium conditions to stathmin/Op18. Inhibition of PP2A in the mitotic extract favour depolymerisation of microtubules, small changes could mimic these effects, so mitotic chromatin may being sufficient to cause a rapid microtubule depolymeri- contain a PP2A inhibitor or a PP2A-activated kinase that sation [11]. It is also possible that sequestration can itself subsequently phosphorylates stathmin/Op18. This is so increase catastrophe frequency, or that a combination of far unresolved, but further establishes a link between both mechanisms is used. stathmin/Op18 and PP2A. The identification of the com- ponents involved in this process is eagerly awaited. Forming the spindle Microtubules become more dynamic during cell division, It was also found that around reflecting breakdown of the interphase network and mitotic chromatin is inhibited by immunodepletion of mitotic spindle function. In this regard, it is interesting stathmin/Op18 from Xenopus egg extracts. This may reflect R214 Current Biology, Vol 8 No 6

a physiologically relevant mechanism for regulating the detect any local changes in phosphorylation. Finally, preferential growth of microtubules around microtubule- interacting drugs, such as taxol, are well- during spindle assembly [14]. The authors emphasise that, known therapeutic agents. Drugs that can modulate the in the system they used, other microtubule-interacting pro- stathmin/Op18–tubulin interaction may therefore be of teins also contribute to spindle formation. As yet, the roles clinical importance. these proteins play and how they cooperate with stath- min/Op18 are not clear; however, they may be regulated in Acknowledgements a similar fashion. I wish to thank A. Sobel and M. Gullberg for providing manuscripts prior to publication. Although studies on mitosis have provided important information on stathmin/Op18 action, it is possible that it References has a somewhat passive role in the regulation of mitosis. 1. Wade RH, Hyman AA: Microtubule structure and dynamics. Curr Opin Cell Biol 1997, 9:12-17. All that may be required is that the microtubule-destabil- 2. McNally FJ: Modulation of microtubule dynamics during the cell ising activity of stathmin/Op18 action is simply switched cycle. Curr Opin Cell Biol 1996, 8:23-29. 3. Sobel A: Stathmin: a relay phosphoprotein for multiple signal off during mitosis. Indeed, stathmin/Op18 is known to be transduction? Trends Biochem Sci 1991, 16:301-305. highly phosphorylated during mitosis. Also, some prolifer- 4. Deacon HW, Mitchison TJ, Gullberg M: Op18/stathmin. In ating cell types do not express stathmin/Op18, and a stath- Guidebook to the Cytoskeletal and Motor Proteins. Oxford: University Press; 1998. min/Op18-deficient knockout mouse develops normally 5. Belmont L, Mitchison T, Deacon HW: Catastrophic revelations [15]. However, this may well be because other stathmin- about Op18/stathmin. Trends Biochem Sci 1996, 21:197-198. related or microtubule-interacting proteins compensate for 6. Belmont LD, Mitchison TJ: Identification of a protein that interacts with tubulin dimers and increases the catastrophe rate of the absence of stathmin/Op18 in these mutant mice. microtubules. Cell 1996, 84:623-631. 7. Marklund U, Larsson N, Melander Gradin H, Brattsand G, Gullberg M: Oncoprotein 18 is a phosphorylation-responsive regulator of A general regulator of microtubules? microtubule dynamics. EMBO J 1996, 15:5290-5298. There is increasing evidence that stathmin/Op18 plays a 8. Horwitz SB, Shen H-J, He L, Dittmar P, Neef R, Chen J, Schubart UK: general role in microtubule regulation in interphase cells, The microtubule destabilising activity of metablastin (p19) is controlled by phosphorylation. J Biol Chem 1997, 272:8129-8132. and it is well established that stathmin/Op18 is phosphory- 9. Larsson N, Marklund U, Melander Gradin H, Brattsand G, Gullberg M: lated in response to diverse extracellular signals [3–5]. Control of microtubule dynamics by oncoprotein 18: dissection of Overexpression of constitutively-active CaMK IV/Gr the regulation of multisite phosphorylation during mitosis. Mol Cell Biol 1997, 17:5530-5539. causes stathmin/Op18 phosphorylation on Ser16 and a con- 10. Curmi PA, Andersen SSL, Lachkar S, Gavet O, Karsenti E, Knossow comitant increase in microtubule mass in the cell [16], thus M, Sobel A: The stathmin/tubulin interaction in vitro. J Biol Chem 1997, 272:25029-25036. linking stathmin/Op18 to calcium signalling. Similar results 11. Jourdain L, Curmi P, Sobel A, Pantaloni D, Carlier M-F: Stathmin: a were obtained with PKA, thereby linking stathmin/Op18 tubulin sequestering protein which forms a ternary T2S complex to cyclic AMP signals [17]. Further support comes from the with two tubulin molecules. Biochemistry 1997, 36:10817-10821. 12. Tournebize R, Andersen SSL, Verde F, Doree M, Karsenti E, Hyman observation that stathmin/Op18 is essential for nerve- AA: Distinct roles of PP1 and PP2A-like phosphatases in control growth-factor-induced differentiation of PC12 cells [18]. of microtubule dynamics during mitosis. EMBO J 1997, 16:5537- 5549. 13. Howell B, Deacon H, Cassimeris L: Injection of against The evidence that stathmin/Op18 is a physiological regula- Op18 results in decreased microtubule catastrophe and tor of microtubule dynamics is quite compelling. However, increased microtubule in vivo. Mol Biol Cell 1997, 8:165a. 14. Andersen SSL, Ashford AJ, Tournebize R, Gavet O, Sobel A, Hyman an in vivo interaction between stathmin/Op18 and tubulin AA, Karsenti E: Mitotic chromatin regulates phosphorylation of has not yet been reported, probably for technical reasons. stathmin/Op18. Nature 1997, 389:640-643. 15. Schubart UK, Yu J, Amat A, Wang Z, Hoffmann MK, Edelman W: SCG10, a stathmin/Op18-related protein expressed in the Normal development of mice lacking metablastin (p19) a , co-purifies with tubulin from calf brain phosphoprotein implicated in cell cycle regulation. J Biol Chem and destabilises microtubules [19]. Further support for an 1996, 271:14062-14066. 16. Melander Gradin H, Marklund U, Larsson N, Chatila TA, Gullberg M: in vivo role of stathmin/Op18 comes from very recent data Regulation of microtubule dynamics by Ca2+/calmodulin showing that lowering stathmin/Op18 levels with an anti- dependent kinase IV/Gr-dependent phosphorylation of body leads to increased tubulin polymerisation, consistent oncoprotein 18. Mol Cell Biol 1997, 17:3459-3467. 17. Melander Gradin H, Larsson N, Marklund U, Gullberg M: Regulation with the proposed function of stathmin/Op18 [13]. of microtubule dynamics by extra-cellular signals: cAMP- dependent protein kinase switches off the activity of oncoprotein 18 in intact cells. J Cell Biol 1998, 140:131-141. Microtubules are also regulated by other proteins [2], 18. Di Paolo G, Pellier V, Catsicas M, Antonsson B, Catsicas S, which probably work together with stathmin/Op18 to give Grenningloh G. The phosphoprotein stathmin is essential for an appropriate response. For example, in interphase much nerve -stimulated differentiation. J Cell Biol 1996, 133:1383-1390. stathmin/Op18 is unphosphorylated but the microtubule 19. Riederer BM, Pellier V, Antonsson B, Di Paolo G, Stimpson SA, network does not collapse, probably because of a counter- Lutjiens R, Catsicas S, Grenningloh G: Regulation of microtubule balancing effect of microtubule-stabilising proteins. dynamics by the removal of growth associated protein SCG10. Proc Natl Acad Sci USA 1997, 94:741-745. Effects on microtubules may occur at specific sub-cellular regions, and phosphospecific antibodies may be useful to