Characterizing the functional impact of novel mutations linked to autism spectrum disorder (ASD) provides a deeper mechanistic understanding of the underlying pathophysiological mechanisms. Here we show that a de novo Glu183 to Val (E183V) mutation in the CaMKII␣ catalytic domain, identified in a proband diagnosed with ASD, decreases both CaMKII␣ substrate phosphorylation and regulatory autophosphorylation, and that the mutated kinase acts in a dominant-negative manner to reduce CaMKII␣-WT autophosphorylation. The E183V mutation also reduces CaMKII␣ binding to established ASD-linked proteins, such as Shank3 and subunits of L-type calcium channels and NMDA receptors, and increases CaMKII␣ turnover in intact cells. In cultured neurons, the E183V mutation reduces CaMKII␣ targeting to dendritic spines. Moreover, neuronal expression of CaMKII␣-E183V increases dendritic arborization and decreases both dendritic spine density and excitatory synaptic transmission. Mice with a knock-in CaMKII␣-E183V mutation have lower total forebrain CaMKII␣ levels, with reduced targeting to synaptic subcellular fractions. The CaMKII␣-E183V mice also display aberrant behavioral phenotypes, including hyperactivity, social interaction deficits, and increased repetitive behaviors. Together, these data suggest that CaMKII␣ plays a previously unappreciated role in ASD-related synaptic and behavioral phenotypes.
The activation of neuronal plasma membrane Ca 2ϩ channels stimulates many intracellular responses. Scaffolding proteins can preferentially couple specific Ca 2ϩ channels to distinct downstream outputs, such as increased gene expression, but the molecular mechanisms that underlie the exquisite specificity of these signaling pathways are incompletely understood. Here, we show that complexes containing CaMKII and Shank3, a postsynaptic scaffolding protein known to interact with L-type calcium channels (LTCCs), can be specifically coimmunoprecipitated from mouse forebrain extracts. Activated purified CaMKII␣ also directly binds Shank3 between residues 829 and 1130. Mutation of Shank3 residues 949 Arg-Arg-Lys 951 to three alanines disrupts CaMKII binding in vitro and CaMKII association with Shank3 in heterologous cells. Our shRNA/rescue studies revealed that Shank3 binding to both CaMKII and LTCCs is important for increased phosphorylation of the nuclear CREB transcription factor and expression of c-Fos induced by depolarization of cultured hippocampal neurons. Thus, this novel CaMKII-Shank3 interaction is essential for the initiation of a specific long-range signal from LTCCs in the plasma membrane to the nucleus that is required for activity-dependent changes in neuronal gene expression during learning and memory.
Neuronal excitation can induce new mRNA transcription, a phenomenon called excitation-transcription (E-T) coupling. Among several pathways implicated in E-T coupling, activation of voltage-gated L-type Ca channels (LTCCs) in the plasma membrane can initiate a signaling pathway that ultimately increases nuclear CREB phosphorylation and, in most cases, expression of immediate early genes. Initiation of this long-range pathway has been shown to require recruitment of Ca-sensitive enzymes to a nanodomain in the immediate vicinity of the LTCC by an unknown mechanism. Here, we show that activated Ca/calmodulin-dependent protein kinase II (CaMKII) strongly interacts with a novel binding motif in the N-terminal domain of Ca1 LTCC α1 subunits that is not conserved in Ca2 or Ca3 voltage-gated Ca channel subunits. Mutations in the Ca1.3 α1 subunit N-terminal domain or in the CaMKII catalytic domain that largely prevent the interaction also disrupt CaMKII association with intact LTCC complexes isolated by immunoprecipitation. Furthermore, these same mutations interfere with E-T coupling in cultured hippocampal neurons. Taken together, our findings define a novel molecular interaction with the neuronal LTCC that is required for the initiation of a long-range signal to the nucleus that is critical for learning and memory.
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