Our recent studies have established that phosphorylation of DARPP-32 and the inhibition of protein phosphatase-1 (PP1) are essential components in mediating the actions of dopamine in the basal ganglia. The disruption of normal dopaminergic neurotransmission, is known to underlie certain neurological diseases, including Huntington's and Parkinson's disease, schizophrenia and attention deficit hyperactivity disorder. The discovery and characterization of basal ganglia phosphoproteins, particular DARPP-32, provides a rational new approach to developing drugs that specifically affect these phosphoproteins or their targets. Our future studies will continue to focus on the detailed biochemical characterization of DARPP-32 and PP1, and on substrates and other regulations of PP1. DARPP-2 is biochemical characterizations of DARPP-32 and PP1, and on substrates and other regulators of PP1. DARPP-32 is phosphorylated at Thr75, by cdk5, converting the protein into an inhibitor of protein kinase A (PKA), and influencing response to activation of PKA by dopamine suggesting a positive feedback mechanism.
In Aim I, we propose to identify he biochemical mechanism(s) involved in the inhibition of PKA by DARPP-32 phosphorylated at Thr75, and to identify the biochemical mechanism(s) involved in the regulation of Thr75 dephosphorylation.
In Aim II, we propose to determine the molecular basis for the interaction of DARPP-32 and PP1. our other studies have identified spinophilin and neurabin, two related F-actin binding proteins that are likely to localized PP1 to specific subcellular compartments, and to modulate PP1 activity towards protein substrates at the targeted sites.
In Aim III, we propose to characterize the phosphorylation and dephosphorylation of spinophilin and neurabin;
in Aim I V, we propose to characterize the interactions between a spinophilin and neurabin, and various binding proteins, including PP1 and F-actin influence of spinophilin and neurabin. In this aim we also propose to identify novel PP1 substrates utilizing mice in which PP1 isoforms have been knocked-out, and using novel mass spectrometric methods. The determination of additional details of the interactions of PP1 with phosphoDARPP32, and with spinophilin and neurabin should prove useful in the development of compounds for the treatment of Parkinson's, schizophrenia and other disorders of dopamine signaling pathways.
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