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.

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).

The meta-cleavage pathway for catechol is one of the major routes for the microbial degradation of aromatic compounds. Pseudomonas sp. strain CF600 grows efficiently on phenol, cresols, and 3,4-dimethylphenol via a plasmid-encoded multicomponent phenol hydroxylase and a subsequent meta-cleavage pathway. The genes for the entire pathway were previously found to be clustered, and the nucleotide sequences of dmpKLMNOPBC and D, which encode the first four biochemical steps of the pathway, were determined. By using a combination of deletion mapping, nucleotide sequence determinations, and polypeptide analysis, we identified the remaining six genes of the pathway. The fifteen genes, encoded in the order dmpKLMNOPQBCDEFGHI, lie in a single operon structure with intergenic spacing that varies between 0 to 70 nucleotides. Homologies found between the newly determined gene sequences and known genes are reported. Enzyme activity assays of deletion derivatives of the operon expressed in Escherichia coli were used to correlate dmpE, G, H, and I with known meta-cleavage enzymes. Although the function of the dmpQ gene product remains unknown, dmpF was found to encode acetaldehyde dehydrogenase (acylating) activity (acetaldehyde:NAD+ oxidoreductase [coenzyme A acylating]; E.C.1.2.1.10). The role of this previously unknown meta-cleavage pathway enzyme is discussed.The central role of catecholic intermediates in aerobic microbial degradation of aromatic compounds is well established. Catechol (1,2-dihydroxybenzene) itself is an intermediate in the degradation of compounds such as benzoate, naphthalene, salicylate, and phenol, and substituted catechols are intermediates in the catabolism of methylated and chlorinated derivatives of these compounds (13,34). A diverse array of enzymes can be elaborated to convert aromatic compounds to central catecholic intermediates. However, the reactions used for oxygenative ring fission of the catechol and the subsequent conversion to Krebs cycle intermediates are limited to one of two metabolic alternatives: those of the ortho-and meta-cleavage pathways. The ortho-cleavage pathways involve ring cleavage between the two hydroxyl groups followed by a well-defined series of reactions leading to P-ketoadipate (reviewed in reference 13). The alternative meta-cleavage pathway involves ring cleavage adjacent to the two catechol hydroxyls, followed by degradation of the ring cleavage product to pyruvate and a short-chain aldehyde (Fig. 1). The use of one pathway or the other is dependent upon the microbial species and/or the nature of the growth substrate.The meta-cleavage pathway was first studied in Pseudomonas strains that can grow at the expense of phenol and cresols (14,29). Since then, the role of the meta-cleavage pathway in aromatic biodegradation by bacteria of many genera, including species of Azotobacter and Alcaligenes and numerous species of Pseudomonas, has been demonstrated (2, 13, 23, 36). In addition, reactions of the lower part of the pathway are involved in the degradation of phenylp...

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.

Oncoprotein 18 (Op18) is a cytosolic protein that was initially identified due to its up‐regulated expression in acute leukemia and its complex pattern of phosphorylation in response to diverse extracellular signals. We have previously identified in vivo phosphorylation sites and some of the protein kinase systems involved. Two distinct proline‐directed kinase families phosphorylate Ser25 and Ser38 of Op18 with overlapping but distinct site preference. These two kinase families, mitogen‐activated protein (MAP) kinases and cyclin‐dependent cdc2 kinases, are involved in receptor‐regulated and cell‐cycle‐regulated phosphorylation events, respectively. During analysis of Op18 phosphorylation in the Jurkat T‐cell line, we also found that Ser16 of Op18 is phosphorylated in response to a Ca2+ signal generated by T‐cell receptor stimulation or the Ca2+ ionophore ionomycin. As suggested by a previous study, T‐cell‐receptor‐induced phosphorylation events may be mediated by the Ca2+/CaM‐dependent protein kinase type Gr (CaM kinase‐Gr). The present study shows that activation of this protein kinase correlates with phosphorylation of Ser16 of Op18, and in vitro experiments reveal efficient and selective phosphorylation of this residue. The CaM kinase‐Gr is only expressed in certain lymphoid cell lines, and the present study shows that ionomycin‐induced phosphorylation of Op18 Ser16 is restricted to cells expressing this protein kinase. Finally, CaM kinase‐Gr‐dependent in vitro phosphorylation of a crude cellular extract reveals a striking preference of this protein kinase for Op18 compared to other cellular substrates. In conclusion, the results suggest that Ser16 of Op18 is a major cytosolic target for activated CaM kinase‐Gr.