Mutations or duplications in MECP2 cause Rett and Rett-like syndromes, neurodevelopmental disorders characterized by mental retardation, motor dysfunction, and autistic behaviors. MeCP2 is expressed in many mammalian tissues and functions as a global repressor of transcription; however, the molecular mechanisms by which MeCP2 dysfunction leads to the neural-specific phenotypes of RTT remain poorly understood. Here, we show that neuronal activity and subsequent calcium influx trigger the de novo phosphorylation of MeCP2 at serine 421 (S421) by a CaMKII-dependent mechanism. MeCP2 S421 phosphorylation is induced selectively in the brain in response to physiological stimuli. Significantly, we find that S421 phosphorylation controls the ability of MeCP2 to regulate dendritic patterning, spine morphogenesis, and the activity-dependent induction of Bdnf transcription. These findings suggest that, by triggering MeCP2 phosphorylation, neuronal activity regulates a program of gene expression that mediates nervous system maturation and that disruption of this process in individuals with mutations in MeCP2 may underlie the neural-specific pathology of RTT.

Neuronal Wiskott-Aldrich Syndrome protein (N-WASP) transmits signals from Cdc42 to the nucleation of actin filaments by Arp2/3 complex. Although full-length N-WASP is a weak activator of Arp2/3 complex, its activity can be enhanced by upstream regulators such as Cdc42 and PI(4,5)P2. We dissected this activation reaction and found that the previously described physical interaction between the NH2-terminal domain and the COOH-terminal effector domain of N-WASP is a regulatory interaction because it can inhibit the actin nucleation activity of the effector domain by occluding the Arp2/3 binding site. This interaction between the NH2- and COOH termini must be intramolecular because in solution N-WASP is a monomer. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) influences the activity of N-WASP through a conserved basic sequence element located near the Cdc42 binding site rather than through the WASp homology domain 1. Like Cdc42, PI(4,5)P2 reduces the affinity between the NH2- and COOH termini of the molecule. The use of a mutant N-WASP molecule lacking this basic stretch allowed us to delineate a signaling pathway in Xenopus extracts leading from PI(4,5)P2 to actin nucleation through Cdc42, N-WASP, and Arp2/3 complex. In this pathway, PI(4,5)P2 serves two functions: first, as an activator of N-WASP; and second, as an indirect activator of Cdc42.

The Arp2/3 complex can be independently activated to initiate actin polymerization by the VCA domain of WASP family members and by the acidic N-terminal and F-actin-binding repeat region of cortactin, which possesses a C-terminal SH3 domain. Cortactin is a target for phosphorylation by Src tyrosine kinases and by serine/threonine kinases that include Erk. Here we demonstrate that cortactin binds N-WASP and WASP via its SH3 domain, induces in vitro N-WASP-mediated actin polymerization, and colocalizes with N-WASP and WASP at sites of active actin polymerization. Erk phosphorylation and a mimicking S405,418D double mutation enhanced cortactin binding and activation of N-WASP. In contrast, Src phosphorylation inhibited the ability of cortactin previously phosphorylated by Erk, and that of S405,418D double mutant cortactin, to bind and activate N-WASP. Furthermore, Y→D mutation of three tyrosine residues targeted by Src (Y421, Y466, and Y482) inhibited the ability of S405,418D cortactin to activate N-WASP. We propose that Erk phosphorylation liberates the SH3 domain of cortactin from intramolecular interactions with proline-rich regions, causing it to synergize with WASP and N-WASP in activating the Arp2/3 complex, and that Src phosphorylation terminates cortactin activation of N-WASP and WASP

The Wiskott-Aldrich syndrome protein (WASP) and its relative neural WASP (N-WASP) regulate the nucleation of actin filaments through their interaction with the Arp2/3 complex and are regulated in turn by binding to GTP-bound Cdc42 and phosphatidylinositol 4,5-bisphosphate. The Nck Src homology (SH) 2/3 adaptor binds via its SH3 domains to a proline-rich region on WASP and N-WASP and has been implicated in recruitment of these proteins to sites of tyrosine phosphorylation. We show here that Nck SH3 domains dramatically stimulate the rate of nucleation of actin filaments by purified N-WASP in the presence of Arp2/3 in vitro. All three Nck SH3 domains are required for maximal activation. Nckstimulated actin nucleation by N-WASP⅐Arp2/3 complexes is further stimulated by phosphatidylinositol 4,5-bisphosphate, but not by GTP-Cdc42, suggesting that Nck and Cdc42 activate N-WASP by redundant mechanisms. These results suggest the existence of an Nck-dependent, Cdc42-independent mechanism to induce actin polymerization at tyrosine-phosphorylated Nck binding sites.