Oncoprotein 18 (Op18, also termed p19, p18, prosolin or stathmin) is a cytosolic protein of previously unknown function. Phosphorylation of Op18 is cell cycle regulated by cyclin‐dependent kinases (CDKs), and expression of a ‘CDK target site‐deficient mutant’ results in a phenotype indicative of a role for Op18 during mitosis. This phenotype is compatible with the idea that Op18 is a phosphorylation‐responsive regulator of microtubule (MT) dynamics. Therefore, in this study, we analyzed MTs in cells induced to express either wild‐type or mutated Op18. The results showed that wild‐type Op18 and a CDK target site mutant both efficiently elicited rapid depolymerization of MTs. This result contrasts with clear‐cut differences in their cell cycle phenotypes. Morphological analysis of MTs explained this apparent discrepancy: while interphase MTs were depolymerized in cells expressing either Op18 derivative, apparently normal mitotic spindles were formed only in cells overexpressing wild‐type Op18. This result correlates with our finding that only mutated Op18 causes a block during mitosis. Hence, we conclude that Op18 decreases MT stability and that this activity of Op18 is subject to cell cycle regulation by CDKs.

Vertebrates express 9–17 septin family members known to oligomerize into diverse structures, but their native assembly states have remained elusive. The results presented suggest a generic model for how the temporal order of septin assembly directs the subunit arrangement within distinct pools of six- to eight-subunit core heteromers.

Oncoprotein 18 (Op18; also termed p19, 19K, metablastin, stathmin, and prosolin) is a conserved protein that regulates microtubule (MT) dynamics. Op18 is multisite phosphorylated on four Ser residues during mitosis; two of these Ser residues, Ser-25 and Ser-38, are targets for cyclin-dependent protein kinases (CDKs), and the other two Ser residues, Ser-16 and Ser-63, are targets for an unidentified protein kinase. Mutations of the two CDK sites have recently been shown to result in a mitotic block caused by destabilization of MTs. To understand the role of Op18 in regulation of MT dynamics during mitosis, in this study we dissected the functions of all four phosphorylation sites of Op18 by combining genetic, morphological, and biochemical analyses. The data show that all four phosphorylation sites are involved in switching off Op18 activity during mitosis, an event that appears to be essential for formation of the spindle during metaphase. However, the mechanisms by which specific sites down-regulate Op18 activity differ. Hence, dual phosphorylation on the CDK sites Ser-25 and Ser-38 appears to be required for phosphorylation of Ser-16 and Ser-63; however, by themselves, the CDK sites are of only minor importance in direct regulation of Op18 activity. Subsequent phosphorylation of either Ser-16, Ser-63, or both efficiently switches off Op18 activity.Microtubules (MTs) are polymeric components of the cytoskeleton found in all eukaryotes and are composed of heterodimers of ␣-and ␤-tubulin. In nondividing cells and during the interphase of the cell cycle, MTs are important for organizing the cytoplasm, for organelle transport, and for intracellular movement of cell surface receptors (for a review, see reference 7). During mitosis, large arrays of MTs, termed the mitotic spindle, segregate the condensed chromosomes (for a review, see reference 17).Tubulin exists in a dynamic equilibrium between free tubulin dimers and MTs. The dynamic instability model describes the dynamic behavior of MTs in terms of the following four parameters: the rates of growth and shrinkage of tubulin polymers and the frequencies of catastrophes (transitions from growth to shrinkage) and rescues (transitions from shrinkage to growth) (30). The dynamic instability of MTs is in part regulated by MT-associated proteins (MAPs), most of which are phosphoproteins (for a review, see reference 15). It is thought that signal transduction cascades that regulate cell proliferation and differentiation control MT dynamics by phosphorylation of MAPs. The consensus in the field is that MAPs stabilize MTs by direct binding and that phosphorylation of MAPs decreases the binding affinity toward MTs and thereby weakens their stabilizing effect (10, 36).

Oncoprotein 18/stathmin (Op18) has been identified recently as a protein that destabilizes microtubules, but the mechanism of destabilization is currently controversial. Based on in vitro microtubule assembly assays, evidence has been presented supporting conflicting destabilization models of either tubulin sequestration or promotion of microtubule catastrophes. We found that Op18 can destabilize microtubules by both of these mechanisms and that these activities can be dissociated by changing pH. At pH 6.8, Op18 slowed microtubule elongation and increased catastrophes at both plus and minus ends, consistent with a tubulin-sequestering activity. In contrast, at pH 7.5, Op18 promoted microtubule catastrophes, particularly at plus ends, with little effect on elongation rates at either microtubule end. Dissociation of tubulin-sequestering and catastrophe-promoting activities of Op18 was further demonstrated by analysis of truncated Op18 derivatives. Lack of a C-terminal region of Op18 (aa 100 -147) resulted in a truncated protein that lost sequestering activity at pH 6.8 but retained catastrophe-promoting activity. In contrast, lack of an N-terminal region of Op18 (aa 5-25) resulted in a truncated protein that still sequestered tubulin at pH 6.8 but was unable to promote catastrophes at pH 7.5. At pH 6.8, both the full length and the N-terminaltruncated Op18 bound tubulin, whereas truncation at the C-terminus resulted in a pronounced decrease in tubulin binding. Based on these results, and a previous study documenting a pH-dependent change in binding affinity between Op18 and tubulin, it is likely that tubulin sequestering observed at lower pH resulted from the relatively tight interaction between Op18 and tubulin and that this tight binding requires the C-terminus of Op18; however, under conditions in which Op18 binds weakly to tubulin (pH 7.5), Op18 stimulated catastrophes without altering tubulin subunit association or dissociation rates, and Op18 did not depolymerize microtubules capped with guanylyl (␣, ␤)-methylene diphosphonatetubulin subunits. We hypothesize that weak binding between Op18 and tubulin results in free Op18, which is available to interact with microtubule ends and thereby promote catastrophes by a mechanism that likely involves GTP hydrolysis.

Oncoprotein 18 (Op18) is a conserved cytosolic protein that is a target for both cell cycle and cell surface receptor-regulated phosphorylation events. The four residues Ser16, Ser25, Ser38, and Ser63 are all subject to cell cycle-regulated phosphorylation. Ser25 and Ser38 are targets for cyclin dependent kinases (CDKs), while Ser16 and Ser63 are phosphorylated by an unidentified protein kinase. We have recently shown that induced expression of a CDK target site-deficient mutant, Op18-S25A,S38A, blocks human cell lines during G2/M transition. In the present report we show that mitosis is associated with complete phosphorylation of the two Op18 CDK target sites Ser25 and Ser38 and that Ser16 and Ser63 are also phosphorylated to a high stoichiometry. To evaluate the function of multisite phosphorylation of Op18, we expressed and analyzed the cell cycle phenotype of different kinase target site-deficient mutants. The data showed that induced expression of the S16A,S63A, S25A,S38A, and S16A,S25A,S38A,S63A mutants all resulted in an indistinguishable phenotype, i.e. immediate G2/M block and subsequent endoreduplication, a given fraction of G2 versus M-phase blocked cells, and a characteristic nuclear morphology of M-blocked cells. This result was unexpected; however, a likely explanation was provided by analysis of Op18 phosphoisomers, which revealed that mutations of the CDK sites interfere with phosphorylation of Ser16 and Ser63. The simplest interpretation of our results is that phosphorylation of Ser16 and Ser63 is essential during G2/M transition and that the phenotype of the S25A,S38A mutant is mediated by the observed block of Ser16/Ser63 phosphorylation.