We will evaluate the role of casein kinase 1 (CK1) and casein kinase 2 (CK2) catalyzed phosphorylation of DARPP-32 and the resultant regulation of protein phosphatase-l (PP-l) in mediating the actions of psychostimulants, morphine and cannabinoids. Drugs of abuse share the ability to enhance dopamine release in nucleus accumbens. Using DARPP-32 KO mice, we have demonstrated that DARPP-32 plays a key role in dopamine signaling and in the actions of drugs of abuse. Activation of dopamine D1 receptors and protein kinase A (PKA) stimulates DARPP-32 phosphorylation at Thr34 and converts DARPP-32 into a PP-1 inhibitor. DARPP-32 is also phosphorylated at Thr75 by cdk5 and thereby converted into a PKA inhibitor. Thus, DARPP-32 is a dual function protein acting either as an inhibitor of PP-l or of PKA. In order to determine the extent to which effects of psychostimulants, morphine and cannabinoids depend upon altered PP-l activity, we will study the actions of these drugs using mutant mice with a point mutation at Thr34. (These studies will complement studies of Thr75 carried out by Project I.) The likelihood of DARPP-32 being phosphorylated at Thr34 depends on the phosphorylation of two serine residues, Ser102 and Ser137. Ser102 and Ser137 are phosphorylated by CK2 and CK1. We have developed phospho-specific antibodies that recognize DARPP-32 when phosphorylated at Ser102 or Ser137. Using these antibodies, we have shown that dopamine alters signaling via casein kinases in striatal slices. We intend to examine the effects of acute and chronic treatment with psychostimulants, morphine and cannabinoids on the state of phosphorylation of DARPP-32 at Ser102 and Ser137. Furthermore, we will also study the actions of drugs of abuse using mutant mice with point mutations at Ser102 or Ser137. These mice, and mice with a point mutation at Thr34, will be characterized with respect to: 1. Phosphorylation of neurotransmitter receptors, and Thr34, Ser102, Ser137 of DARPP-32; 2. Electrophysiological parameters in dissociated neurons and rain slices; 3. Gene transcription using in situ hybridization and gene chip technology, with altered gene products being analyzed with respect to their distribution, phosphorylation and interactions with other components of the dopamine signaling pathway; 4. Locomotion, place preference, self-administration and escalation of drug intake. Taken together, these studies will provide detailed information on the involvement of the DARPP-32/PP-l signaling cascade in the actions of drugs of abuse and whether CKl and CK2 may be novel targets for treatment of drug abuse.
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