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The microtubule-destabilizing activity of metablastin (p19) is controlled by phosphorylation
1Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
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 serine-to-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.
Insights
Metablastin, a protein regulating microtubules, destabilizes them in cells and in vitro. Phosphorylation controls its activity in vivo, suggesting a role in cell division and development.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Metablastin (p19/stathmin) is a conserved protein in immature vertebrate cells.
- It is regulated by extracellular factors and the cell cycle through serine phosphorylation.
- Previous studies showed metablastin destabilizes microtubules in vitro.
Purpose of the Study:
- To investigate the role of metablastin in microtubule dynamics within living cells.
- To determine the impact of phosphorylation site mutations on metablastin's activity.
- To elucidate the mechanism by which metablastin affects microtubules.
Main Methods:
- Microinjection of recombinant metablastin into COS-7 cells.
- Site-directed mutagenesis of phosphorylation sites (Ser-to-Ala and Ser-to-Asp).
- In vitro tubulin assembly assays.
Main Results:
- Microinjection of metablastin caused microtubule loss in COS-7 cells.
- Serine-to-alanine mutations enhanced microtubule destabilization, while Ser-63 aspartate substitution abolished it.
- Metablastin stoichiometrically inhibited microtubule assembly and promoted disassembly in vitro.
- In vitro activity was unaffected by phosphorylation site mutations, unlike in vivo effects.
Conclusions:
- Metablastin phosphorylation regulates its microtubule-destabilizing activity in vivo.
- This regulation likely requires additional cellular factors.
- Metablastin plays a key role in microtubule reorganization during morphogenesis and mitosis.