Protein Phosphatase Inhibitor-1 and Cdk5: Of Molecules and Memory
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Protein phosphatase inhibitor-1 and cyclin-dependent kinase 5 (Cdk5) have been independently implicated in synaptic plasticity, learning, and memory. We began our studies with the identification, confirmation, and characterization of a novel Cdk5-dependent phosphorylation site (Ser6) on inhibitor-1. In the striatum, basal in vivo phosphorylation and dephosphorylation of Ser6 were mediated by Cdk5 and protein phosphatases 2A (PP-2A) and 1 (PP-1), respectively. Additionally, protein phosphatase 2B (PP-2B) contributed to dephosphorylation under conditions of high Ca2+. Functionally, Cdk5-dependent phosphorylation of inhibitor-1 intramolecularly impaired dephosphorylation and deactivation of the protein, placing the activities of Cdk5 and protein kinase A (PKA) in synergism in the negative regulation of PP-1. These studies uncovered a potential new regulatory mechanism for Cdk5. Investigation revealed that depolarization differentially regulates the Cdk5-dependent sites of inhibitor-1 and its homologue dopamine- and cAMP-regulated phosphoprotein (DARPP-32) in a cofactor- and N-methyl-D-aspartate (NMDA) receptor-independent manner. Effects on DARPP-32 were Ca2+-mediated and PP-2A-dependent, while effects on inhibitor-1 were nonselectively cation-mediated and either partially PP-2B-dependent or independent of the major serine/threonine phosphatases, depending on the site. Given the uncertain role of inhibitor-1 in learning and memory, we next focused on identifying behaviors and substrates impacted by inhibitor-1 function. Mice constitutively lacking inhibitor-1 displayed enhanced neurogenesis and mildly impaired habituation, but normal contextual fear and novelty learning. Furthermore, levels of hippocampal inhibitor-1 were increased by voluntary wheel running, a stimulus for neurogenesis. Thus, inhibitor-1 may function in an anti-neurogenic mechanism and be more important in the direct or indirect modulation of dopamine-dependent behaviors than in the mnemonic functions of the hippocampus. Using a whole-cell patch clamp approach, we also attempted to identify electrical properties of dentate granule cells that might be affected by Cdk5-dependent phosphorylation of inhibitor-1. Most promising among the results was a reduction in the ability of granule cells lacking inhibitor-1 to faithfully respond to high-frequency trains of stimuli. Granule cell excitability was also increased by pharmacological inhibition of Cdk5 with roscovitine. Finally, in a related study, we helped firmly establish a role for Cdk5 in hippocampal synaptic plasticity by demonstrating that conditional loss of Cdk5 enhances NMDA receptor-mediated currents, particularly of the NR2B type.