Abstract
Spinophilin is a protein phosphatase 1 (PP1)- and actin-binding protein that modulates excitatory synaptic transmission and dendritic spine morphology. We report that spinophilin is phosphorylated in vitro by protein kinase A (PKA). Phosphorylation of spinophilin was stimulated by treatment of neostriatal neurons with a dopamine D1 receptor agonist or with forskolin, consistent with spinophilin being a substrate for PKA in intact cells. Using tryptic phosphopeptide mapping, site-directed mutagenesis, and microsequencing analysis, we identified two major sites of phosphorylation, Ser-94 and Ser-177, that are located within the actin-binding domain of spinophilin. Phosphorylation of spinophilin by PKA modulated the association between spinophilin and the actin cytoskeleton. Following subcellular fractionation, unphosphorylated spinophilin was enriched in the postsynaptic density, whereas a pool of phosphorylated spinophilin was found in the cytosol. F-actin co-sedimentation and overlay analysis revealed that phosphorylation of spinophilin reduced the stoichiometry of the spinophilin-actin interaction. In contrast, the ability of spinophilin to bind to PP1 remained unchanged. Taken together, our studies suggest that phosphorylation of spinophilin by PKA modulates the anchoring of the spinophilin-PP1 complex within dendritic spines, thereby likely contributing to the efficacy and plasticity of synaptic transmission.
Highlights
Dendritic spines are specialized protrusions that receive the majority of excitatory input in the central nervous system [1,2,3]
Our studies suggest that phosphorylation of spinophilin by protein kinase A (PKA) modulates the anchoring of the spinophilin-phosphatase 1 (PP1) complex within dendritic spines, thereby likely contributing to the efficacy and plasticity of synaptic transmission
Two-dimensional phosphopeptide maps of spinophilin phosphorylated by PKA to low (1-min incubation) or higher (60-min incubation) stoichiometry revealed the presence of five major phosphopeptides
Summary
Our studies suggest that phosphorylation of spinophilin by PKA modulates the anchoring of the spinophilin-PP1 complex within dendritic spines, thereby likely contributing to the efficacy and plasticity of synaptic transmission. Spinophilin is likely to influence the dynamic behavior of dendritic spines by its ability to modulate the actin cytoskeleton. Cultured neurons from spinophilin knockout mice had more filopodia, or spine-like protrusions, but the same number of nerve terminals as wild-type mice These observations suggest that spinophilin may either facilitate spine retraction or suppress the initial outgrowth of spines from the dendrite. Spinophilin may function as a scaffold protein to regulate cross-talk between various physiological stimuli in dendritic spines It remains to be determined whether spinophilin is regulated by specific neurotransmitter systems in the brain. Studied the functional consequences of spinophilin phosphorylation on its interactions with actin and PP1
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