Role of Ser50 phosphorylation in SCG10 regulation of microtubule depolymerization.
Members of the stathmin-like protein family depolymerize microtubules (MTs), probably due to the ability of each stathmin monomer to bind two tubulin heterodimers in a complex (T(2)S complex). SCG10, a member of this family, is localized in the growth cone of neurons. It has four identified sites of serine phosphorylation (S50, S63, S73, and S97). Of these, S50 and S97 are phosphorylated by cAMP-dependent protein kinase, an enzyme involved in growth cone guidance. When the equivalent sites in stathmins are phosphorylated, they lose their ability to depolymerize MTs. We investigated the specific role of the two cAMP-dependent protein kinase (PKA) phosphorylation sites in SCG10. A mutant of SCG10 phosphorylated only on S50 retained the ability to depolymerize MTs, but SCG10 phosphorylated on S97 or on both S50 and S97 lost MT-depolymerizing activity. Surface plasmon resonance studies revealed that the phosphorylation of SCG10 at these sites reduced the tubulin heterodimer binding, mainly due to a reduced rate of association. In particular, compared to the two other phosphorylated forms, SCG10 phosphorylated at S50 had a significantly smaller dissociation constant for the binding of the first tubulin heterodimer and larger association and dissociation rate constants for the binding of the second heterodimer. This indicates that the phosphorylation of S50 compensates for the effect of phosphorylation at other sites by modulating T2S complex formation. Furthermore, these results suggest that S50-P maintains MT-depolymerizing activity, which indicates that the biological functions of phosphorylation at S50 and S97 are different.
Togano
Tetsuya
T
Division of Molecular and Cellular Biology, Department of Signal Transduction Research, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan.
Kurachi
Masashi
M
Watanabe
Michitoshi
M
Grenningloh
Gabriele
G
Igarashi
Michihiro
M
D000818:Animals; D002352:Carrier Proteins; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D066298:In Vitro Techniques; D008565:Membrane Proteins; D008868:Microtubule Proteins; D008870:Microtubules / Q000378:metabolism*; D009414:Nerve Growth Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion / Q000502:physiology; D012694:Serine / Q000378:metabolism*; D020349:Surface Plasmon Resonance / Q000379:methods; D013997:Time Factors; D014404:Tubulin / Q000378:metabolism / Q000494:pharmacology
D002352:Carrier Proteins; D008565:Membrane Proteins; D008868:Microtubule Proteins; D009414:Nerve Growth Factors; D011994:Recombinant Proteins; C512848:Stmn2 protein, rat; D014404:Tubulin; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases