The Dopamine/D1 Receptor Mediates the Phosphorylation and Inactivation of the Protein Tyrosine Phosphatase STEP via a PKA-Dependent Pathway

The striatal-enriched protein tyrosine phosphatase (STEP) family is expressed within dopaminoceptive neurons of the CNS and is particularly enriched within the basal ganglia and related structures. Alternative splicing produces several isoforms that are found in a number of subcellular compartments, including postsynaptic densities of medium spiny neurons. The variants include STEP61, a membrane-associated protein, and STEP46, a cytosolic protein. The C terminals of these two isoforms are identical, whereas the N-terminal domain of STEP61 contains a novel 172 amino acid sequence that includes several structural motifs not present in STEP46. Amino acid sequencing revealed a number of potential phosphorylation sites in both STEP isoforms. Therefore, we investigated the role of phosphorylation in regulating STEP activity. Both STEP61 and STEP46 are phosphorylated on seryl residues by a cAMP-dependent protein kinase (PKA)-mediated pathway in striatal homogenates. The specific residues phosphorylated in STEP61 were identified by site-directed mutagenesis and tryptic phosphopeptide mapping as Ser160 and Ser221, whereas the major site of phosphorylation in STEP46 was shown to be Ser49. Ser160 is located within the unique N terminal of STEP61. Ser221 and Ser49 are equivalent residues present in STEP61 and STEP46, respectively, and are located at the center of the kinase-interacting motif that has been implicated in protein–protein interactions. Phosphorylation at this site decreases the activity of STEP in vitro by reducing its affinity for its substrate. In vivo studies using striatal slices demonstrated that the neurotransmitter dopamine leads to the phosphorylation of STEP via activation of D1 receptors and PKA.

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