The Microtubule-destabilizing Activity of Metablastin (p19) Is Controlled by Phosphorylation

Abstract

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...

Overexpression of the stathmin gene in a subset of human breast cancer

“…To account for these effects, we demonstrated that stathmin directly interacts with, and sequesters, tubulin (Curmi et al, 1997) in a T2S complex (Jourdain et al, 1997), and that this sequestration leads to the displacement of the microtubule/tubulin equilibrium towards depolymerization of microtubules (Jourdain et al, 1997). Importantly, phosphorylation of stathmin altered the affinity of stathmin for tubulin (Marklund et al, 1996;Curmi et al, 1997;Di Paolo et al, 1997;Horwitz et al, 1997;Larsson et al, 1997). Together, these results give insight into the observed physiological variations of stathmin phosphorylation during the mitotic cycle (Strahler et al, 1992;Brattsand et al, 1994) and argue for the search for stathmin dysregulations in tumours as well as an understanding of its mechanisms.…”

“…We demonstrated that this phenomenon is related to a direct interaction of stathmin with tubulin dimers, leading to the sequestration of tubulin in a twotubulin heterodimer-one-stathmin complex (T2S) (Curmi et al, 1997;Jourdain et al, 1997). Furthermore, it has been shown that phosphorylation of stathmin dramatically reduces its affinity for tubulin and its microtubule-destabilizing activity (Marklund et al, 1996;Curmi et al, 1997;Di Paolo et al, 1997;Horwitz et al, 1997;Larsson et al, 1997), giving an additional clue to the mechanisms of the in vivo control of microtubule reorganization during mitosis. Finally, the stathmin gene maps to lp35-36.1 (Ferrari et al, 1990), in a region (lp32-lpter) thought to harbour at least one tumoursuppressor gene (Bieche et al, 1994).…”

Mitogen-activated protein kinase pathway is dispensable for microtubule-active drug-induced Raf-1/Bcl-2 phosphorylation and apoptosis in leukemia cells

“…56,57 Microtubules are associated with serine/threonine kinases and phosphatases. [58][59][60][61] As the plasma membrane is an important source of signals during interphase, microtubules may be a principal location of signaling following mitotic arrest.…”

Overexpression of stathmin1 in the diffuse type of gastric cancer and its roles in proliferation and migration of gastric cancer cells

“…Its expression in cancer cells has been associated with their proliferation and metastasis (Mistry and Atweh, 2006;Singer et al, 2007). Depending on its phosphorylation state, which is mediated by p34cdc2 kinase, mitogen-activated protein kinase and other kinases, stathmin1 can regulate cell cycle through modulation of the mitotic spindle (Marklund et al, 1993(Marklund et al, , 1996Luo et al, 1994;Larsson et al, 1995;Horwitz et al, 1997;Mistry and Atweh, 2006). Interestingly, when biopsy specimens from human prostate cancers were immunostained with an anti-stathmin1 antibody, immunoreactivity was seen in poorly differentiated tumours but not in hyperplastic prostate or highly differentiated tumours (Friedrich et al, 1995;Mistry and Atweh, 2006).…”