Abstract
Stathmin is an important regulator of microtubule polymerization and dynamics. When unphosphorylated it destabilizes microtubules in two ways, by reducing the microtubule polymer mass through sequestration of soluble tubulin into an assembly-incompetent T2S complex (two alpha:beta tubulin dimers per molecule of stathmin), and by increasing the switching frequency (catastrophe frequency) from growth to shortening at plus and minus ends by binding directly to the microtubules. Phosphorylation of stathmin on one or more of its four serine residues (Ser(16), Ser(25), Ser(38), and Ser(63)) reduces its microtubule-destabilizing activity. However, the effects of phosphorylation of the individual serine residues of stathmin on microtubule dynamic instability have not been investigated systematically. Here we analyzed the effects of stathmin singly phosphorylated at Ser(16) or Ser(63), and doubly phosphorylated at Ser(25) and Ser(38), on its ability to modulate microtubule dynamic instability at steady-state in vitro. Phosphorylation at either Ser(16) or Ser(63) strongly reduced or abolished the ability of stathmin to bind to and sequester soluble tubulin and its ability to act as a catastrophe factor by directly binding to the microtubules. In contrast, double phosphorylation of Ser(25) and Ser(38) did not affect the binding of stathmin to tubulin or microtubules or its catastrophe-promoting activity. Our results indicate that the effects of stathmin on dynamic instability are strongly but differently attenuated by phosphorylation at Ser(16) and Ser(63) and support the hypothesis that selective targeting by Ser(16)-specific or Ser(63)-specific kinases provides complimentary mechanisms for regulating microtubule function.
Highlights
Stathmin is an 18-kDa ubiquitously expressed microtubuledestabilizing phosphoprotein whose activity is modulated by phosphorylation of its four serine residues, Ser[16], Ser[25], Ser[38], and Ser[63] (1–7)
Sequestration of tubulin into T2S complexes destabilizes microtubules by reducing the quantity of assembly-competent tubulin and by increasing indirectly the catastrophe frequency as the microtubules depolymerize to a new steady state
We analyzed the effects of stathmin on dynamic instability after the microtubules attained steady state; a condition at which a stable steady-state equilibrium between polymerized microtubules and soluble tubulin is maintained
Summary
Stathmin is an 18-kDa ubiquitously expressed microtubuledestabilizing phosphoprotein whose activity is modulated by phosphorylation of its four serine residues, Ser[16], Ser[25], Ser[38], and Ser[63] (1–7). Consistent with its strong catastrophe-promoting activity at minus ends, stathmin increases the treadmilling rate of steady-state microtubules in vitro (27). Phosphorylation of Ser[16] or Ser[63] appears to be more critical than phosphorylation of Ser[25] and Ser[38] for the ability of stathmin to bind to soluble tubulin and to inhibit microtubule assembly in vitro (3, 25). Inhibition of stathmin phosphorylation induces defects in spindle assembly and organization (3, 14) suggesting that soluble tubulin-microtubule levels are regulated by phosphorylation of stathmin, but the dynamics of microtubules could be regulated in a phosphorylation-dependent manner It is not known how phosphorylation at any of the four serine residues of stathmin affects its ability to regulate microtubule dynamics and, its ability to increase the catastrophe frequency at plus and minus ends due to its direct interaction with microtubules. The data further support the hypotheses that phosphorylation of stathmin on either Ser[16] or Ser[63] plays a critical role in regulating microtubule polymerization and dynamics in cells
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