Phosphorylation of Microtubule-associated Protein STOP by Calmodulin Kinase II

Julie Baratier,Leticia Peris,Jacques Brocard,Sylvie Gory-Fauré,Fabrice Dufour,Christophe Bosc,Anne Fourest-Lieuvin,Laurent Blanchoin,Paul Salin,Didier Job,Annie Andrieux

doi:10.1074/jbc.m509602200

Abstract

STOP proteins are microtubule-associated, calmodulin-regulated proteins responsible for the high degree of stabilization displayed by neuronal microtubules. STOP suppression in mice induces synaptic defects affecting both short and long term synaptic plasticity in hippocampal neurons. Interestingly, STOP has been identified as a component of synaptic structures in neurons, despite the absence of microtubules in nerve terminals, indicating the existence of mechanisms able to induce a translocation of STOP from microtubules to synaptic compartments. Here we have tested STOP phosphorylation as a candidate mechanism for STOP relocalization. We show that, both in vitro and in vivo, STOP is phosphorylated by the multifunctional enzyme calcium/calmodulin-dependent protein kinase II (CaMKII), which is a key enzyme for synaptic plasticity. This phosphorylation occurs on at least two independent sites. Phosphorylated forms of STOP do not bind microtubules in vitro and do not co-localize with microtubules in cultured differentiating neurons. Instead, phosphorylated STOP co-localizes with actin assemblies along neurites or at branching points. Correlatively, we find that STOP binds to actin in vitro. Finally, in differentiated neurons, phosphorylated STOP co-localizes with clusters of synaptic proteins, whereas unphosphorylated STOP does not. Thus, STOP phosphorylation by CaMKII may promote STOP translocation from microtubules to synaptic compartments where it may interact with actin, which could be important for STOP function in synaptic plasticity.

Highlights

Actin binding assay was performed with CE fraction containing 0.5 ␮M phospho- or dephospho-STOP (as estimated on Coomassie Blue gels, using known amounts of pure STOP as standards) complemented or not with various amount of G-actin (1, 6, and 15 ␮M), in 100 ␮l of a 1ϫ F-polymerization buffer (10ϫ stock: 50 mM Tris-HCl, 5 mM dithiothreitol, 5 mM ATP, 1 M KCl, 50 mM MgCl2, pH 7.5) in the presence of 1 mM EGTA
Phosphorylated N-STOP Does Not Bind to Microtubules in Vitro—We investigated whether the phosphorylation of STOP by calmodulin-dependent protein kinase II (CaMKII) could affect STOP binding to microtubules, as phosphorylation often regulates the binding on microtubules of associated proteins [16, 17]
STOP proteins are important for synaptic plasticity, as demonstrated by the synaptic defects in both long and short term plasticity exhibited by STOP-deficient mice [7]

Summary

Introduction

Actin binding assay was performed with CE fraction containing 0.5 ␮M phospho- or dephospho-STOP (as estimated on Coomassie Blue gels, using known amounts of pure STOP as standards) complemented or not with various amount of G-actin (1, 6, and 15 ␮M), in 100 ␮l of a 1ϫ F-polymerization buffer (10ϫ stock: 50 mM Tris-HCl, 5 mM dithiothreitol, 5 mM ATP, 1 M KCl, 50 mM MgCl2, pH 7.5) in the presence of 1 mM EGTA. Incubation of CE fraction proteins with [␥-32P]ATP in the absence of Ca2ϩ-calmodulin followed by SDS-PAGE analysis and autoradiography showed little phosphate incorporation in the bands corresponding to N-STOP or to CaMKII (Fig. 1E, left column).

Results

Conclusion