Phosphorylation-mediated regulation of Arc interactions with the AMPA receptor endocytic machinery - PROJECT SUMMARY The activity-regulated cytoskeletal-associated protein (Arc, also known as Arg3.1) is an immediate early gene product induced by activity/experience and required for multiple modes of synaptic plasticity, including long-term potentiation (LTP), long-term depression (LTD), and homeostatic scaling. It has been implicated in functional and morphological changes in dendritic spines that underlie learning and memory. The best-characterized function of Arc is enhancement of the endocytic internalization of AMPA receptors (AMPARs), a process associated with LTD. Aside from studies showing that Arc binds directly to three elements of the endocytic machinery: the membrane endophilin, dynamin and AP-2, almost nothing is known about the mechanism or regulation of Arc function in AMPAR internalization. We previously reported that Arc palmitoylation is important for mGluR-mediated synaptic weakening, and more recently found that phosphorylation by PKC blocks this modification, suggesting that PKC phosphorylation may serve to limit LTD and/or to enhance LTP. The two PKC phosphorylated serines lie within the endophilin binding motif, raising the possibility that their phosphorylation inhibits the Arc-endophilin interaction. Arc is phosphorylated by the MAP kinase ERK on a serine located within the dynamin and AP-2 binding motifs, again suggesting an inhibitory role for this modification. We recently observed that Arc is an excellent substrate for TAOK2, a kinase that stabilizes the postsynaptic density scaffolding protein PSD95. TAOK2 phosphorylates Arc within its C-terminal domain, the site of interaction with the AMPAR auxiliary subunit, stargazin. Work from others demonstrated that Arc competes with PSD95 for binding to stargazin, suggesting a model in which dissociation of stargazin from PSD95 and association of stargazin with Arc contributes to the delivery of Arc from the PSD to endocytic zones within dendritic spines. We hypothesize that Arc interactions with endophilin, dynamin, AP-2, and stargazin are inhibited upon phosphorylation of Arc by PKC, ERK, and/or TAOK2. In Aims 1 and 2 of this proposal we will use biochemical and biophysical methods and fluorescence-based nanoimaging approaches to characterize the interactions of Arc with endophilin, dynamin, AP-2, and stargazin in vitro and in living heterologous cells. Effects of phosphorylation by PKC, ERK, and TAOK2 on these interactions will be assessed. Based on information obtained in Aims 1 and 2, a subset of interactions and phosphorylation site mutations will be investigated in hippocampal neurons from Arc knockout mice. Synaptic weakening will be examined electrophysiologically and AMPAR internalization will be monitored using immunohistochemical and surface biotinylation assays. We will also measure changes in Arc phosphorylation at sites phosphorylated by the three kinases in response to LTP- and LTD-inducing protocols. Increases and decreases in Arc expression have been linked to numerous cognitive disorders, including Fragile X syndrome, Alzheimer’s disease, and substance abuse. Therefore, we anticipate that elucidation of novel mechanisms of Arc regulation will have therapeutic significance.
