Regulation of microtubule dynamic instability in vitro by differentially phosphorylated stathmin.
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.
Manna
Tapas
T
Department of Molecular, Cellular, and Developmental Biology and the Neuroscience Research Institute, University of California, Santa Barbara, California 93106, USA.
Thrower
Douglas A
DA
Honnappa
Srinivas
S
Steinmetz
Michel O
MO
Wilson
Leslie
L
D000409:Alanine / Q000378:metabolism; D000818:Animals; D054468:Axoneme / Q000187:drug effects / Q000502:physiology; D007700:Kinetics; D018715:Microscopy, Video; D008870:Microtubules / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011489:Protein Denaturation; D020673:Protein Renaturation; D012617:Sea Urchins; D050777:Stathmin / Q000378:metabolism / Q000494:pharmacology*; D014404:Tubulin / Q000187:drug effects / Q000378:metabolism
D050777:Stathmin; D014404:Tubulin; D010768:Phosphoserine; D000409:Alanine