Phosphorylation of Microtubule-associated Protein STOP by Calmodulin Kinase II

Abstract

STOP proteins are microtubule-associated, calmodulinregulated 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.

Structural Basis for the Association of MAP6 Protein with Microtubules and Its Regulation by Calmodulin

“…This may explain previous reports showing an unusual binding mode for these CaM-binding sites (22). 5 Interestingly, some 1-5-10 motifs were shown to be implicated both in Ca 2ϩ -CaM and actin binding (47)(48)(49), and MAP6-N was shown to interact with actin (40). It would be of interest to probe whether this region is implicated in MAP6-N association with actin and whether Ca 2ϩ -CaM binding to this region may regulate such process.…”

“…Association of -calmodulin-dependent protein kinase II, which triggers the translocation of MAP6 from MTs to actin (40). Interestingly, this kind of regulation has already been observed for the microtubule-associated protein p35, which is also regulated by site-specific phosphorylations and Ca 2ϩ -CaM with MT overlapping binding sites (41).…”

Microtubule Organization and Microtubule-Associated Proteins (MAPs)

“…3.1) (Guillaud et al 1998). MAP6 is also present in dendrites and there are some evidences that localize MAP6 to synapses in mature hippocampal neurons (Andrieux et al 2002;Baratier et al 2006;Peng et al 2004). CaMKII phosphorylation may promote MAP6 translocation from MTs to synaptic compartments where it interacts with actin.…”

“…CaMKII phosphorylation may promote MAP6 translocation from MTs to synaptic compartments where it interacts with actin. This translocation could be important during synaptic plasticity (Baratier et al 2006). MAP6 is also important for neurite formation and dendritic arborization, where it has been suggested to act as a molecular brake for lysosomal trafficking in dendrites (Guillaud et al 1998;Schwenk et al 2014).…”

“…MAP6 also includes four consensus sites for phosphorylation by CaM kinase II that regulates its binding to MTs (Bosc et al 2003). MAP6 can also bind actin (Baratier et al 2006). Phosphorylated forms of MAP6 cannot bind MTs and co-localize with actin along the neurites and at branching points.…”