Metablastin (also called p19, stathmin, prosolin, p18, Lap18, and oncoprotein 18) is a highly conserved, cytosolic 149-amino acid polypeptide that is expressed in immature vertebrate cells and undergoes extracellular factor-and cell cycle-regulated serine phosphorylation. The protein was shown recently to destabilize microtubules in vitro (Belmont, L., and Mitchison, T. J. (1996) Cell 84, 623-631). Here we demonstrate that microinjection of recombinant metablastin induces a loss of microtubules in COS-7 cells. This effect is enhanced by serineto-alanine mutations at several phosphorylation sites and virtually abolished by aspartate substitution at a single site, Ser-63. We also show that stoichiometric amounts of metablastin prevent assembly and promote disassembly of microtubules in vitro. Interestingly, the phosphorylation site mutations of metablastin that have dramatic differential effects in intact cells do not alter the ability of metablastin to block tubulin assembly in vitro. The data suggest that phosphorylation of metablastin controls its microtubule-destabilizing activity in vivo but that this regulation may require additional cellular factors. This control mechanism is poised to play a critical role in the dynamic reorganization of the cellular microtubule network that occurs during morphogenesis and mitosis.Serine phosphorylation of metablastin is stimulated in mammalian cells by a diverse group of extracellular factors, which include cAMP-linked agonists (1-3), factors known to activate protein kinase C (3, 4), growth factors that initiate signaling through tyrosine kinase receptors (5, 6), heat shock (7), and, in some cells, agents that induce calcium flux (8). Furthermore, the phosphorylation state of metablastin fluctuates during the cell cycle, achieving its highest level in M phase (9 -11).The known phosphorylation sites of metablastin are . Although the specific protein kinases that directly phosphorylate metablastin in vivo have not been identified, the available evidence suggests that, in cells, Ser-63 and, to a lesser extent, Ser-16 are phosphorylated by cAMP-dependent protein kinase (14), Ser-25 and Ser-38 by mitogen-activated protein kinase(s) and cyclin-dependent kinase(s) (14 -16), and Ser-16 by Ca 2ϩ /calmodulin kinase-GR (8).To test the microtubule-destabilizing activity of metablastin in intact cells and to explore the potential role of phosphorylation in regulating this activity, we have introduced recombinant metablastin and phosphorylation site mutants into COS-7 cells by microinjection and assessed changes in the cellular microtubule array by immunocytochemistry.
EXPERIMENTAL PROCEDURESPreparation of Recombinant Proteins-Metablastin and mutated forms of the protein were expressed as GST 1 fusion proteins in Escherichia coli. Metablastin cDNA (17) encoding amino acids 6 -145 was amplified using the polymerase chain reaction (forward primer, 5Ј-AGGGATCCAGGTGAAAGAGCTGGAGAAG-3Ј; reverse primer, 5ЈCA-AAGACCCCGCGGACGAGAATTCCA3Ј; 30 temperature cycles: 94°C, 1 min; 60°C, 45 s; 72°C, 30...