Phosphorylation of DARPP-32 by Cdk5 modulates dopamine signalling in neurons

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Abstract

The physiological state of the cell is controlled by signal transduction mechanisms which regulate the balance between protein kinase and protein phosphatase activities1. Here we report that a single protein can, depending on which particular amino-acid residue is phosphorylated, function either as a kinase or phosphatase inhibitor. DARPP-32 (dopamine and cyclic AMP-regulated phospho-protein, relative molecular mass 32,000) is converted into an inhibitor of protein phosphatase 1 when it is phosphorylated by protein kinase A (PKA) at threonine 34 (refs 2, 3). We find that DARPP-32 is converted into an inhibitor of PKA when phosphorylated at threonine 75 by cyclin-dependent kinase 5 (Cdk5). Cdk5 phosphorylates DARPP-32 in vitro and in intact brain cells. Phospho-Thr 75 DARPP-32 inhibits PKA in vitro by a competitive mechanism. Decreasing phospho-Thr 75 DARPP-32 in striatal slices, either by a Cdk5-specific inhibitor or by using genetically altered mice, results in increased dopamine-induced phosphorylation of PKA substrates and augmented peak voltage-gated calcium currents. Thus DARPP-32 is a bifunctional signal transduction molecule which, by distinct mechanisms, controls a serine/threonine kinase and a serine/threonine phosphatase.

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Figure 1: Phosphorylation of DARPP-32 at Thr 75 by Cdk5 in vitro and in vivo.
Figure 2: Inhibition of PKA by phospho-Thr 75 DARPP-32.
Figure 3: Effect of phospho-Thr 75 DARPP-32 on PKA activity in intact neurons.
Figure 4: Model illustrating dual effects of phosphorylation of DARPP-32 in regulation of PKA and PP1 activities.

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Acknowledgements

We thank A. Horiuchi for construction of the Thr75A mutant plasmid; S. Rakhilin for recombinant ARPP-21; I. Dulubova for phospho-Ser88 ARPP-16 antibody; J. Wang for recombinant Cdk5/p25; J. Volker for help in providing p35-/- mice; and the Rockefeller University Protein/DNA Technology Center. This work was supported by a National Research Service Award (J.B.) and funding from the National Institute of Mental Health and the National Institute of Drug Abuse (P.G.).

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Correspondence to Paul Greengard.

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Bibb, J., Snyder, G., Nishi, A. et al. Phosphorylation of DARPP-32 by Cdk5 modulates dopamine signalling in neurons. Nature 402, 669–671 (1999) doi:10.1038/45251

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