Neuronal L-Type Calcium Channel Signaling to the Nucleus Requires a Novel CaMKIIα-Shank3 Interaction

Abstract

The molecular mechanisms that couple plasma membrane receptors/channels to specific intracellular responses, such as increased gene expression, are incompletely understood. The postsynaptic scaffolding protein Shank3 associates with Ca 2+ permeable receptors or ion channels that can activate many downstream signaling proteins, including calcium/calmodulin-dependent protein kinase II (CaMKII). Here, we show that Shank3/CaMKIIa complexes can be specifically co-immunoprecipitated from mouse forebrain lysates, and that purified activated (Thr286 autophosphorylated) CaMKIIa binds directly to Shank3 between residues 829-1130. Mutation of three basic residues in Shank3 (R 949 RK 951 ) to alanine disrupts CaMKII binding to Shank3 fragments in vitro, as well as CaMKII association with full-length Shank3 in heterologous cells. Our shRNA/rescue studies revealed that Shank3 binding to both CaMKII and L-type calcium channels (LTCCs) is required for increased phosphorylation of the nuclear CREB transcription factor induced by depolarization of cultured hippocampal neurons. Thus, this novel Shank3-CaMKII interaction is essential for the initiation of a specific long-range signal from plasma membrane LTCCs to the nucleus that is required for activitydependent changes in neuronal gene expression during learning and memory.

CaMKII Phosphorylation Regulates Synaptic Enrichment of Shank3

“…CaMKII are coimmunoprecipitated with HA-Shank3 as shown previously (Perfitt et al, 2020b), but the １１ pS782 immunoreactivity for HA-Shank3 was specifically increased upon active CaMKII cotransfection, which was not the case for the inactive CaMKII R42K cotransfection (Fig. 2D).…”

“…2D). Notably, the CaMKII binding motif, R -R 950 -K 951 (Perfitt et al, 2020b) has been characterized and the Shank3 S782 phosphorylation site is not far from the binding motif, further implicating CaMKII regulation ( Fig. 2A).…”