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

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). https://doi.org/10.1038/45251

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