Regulation of protein phosphatase inhibitor-1 by cyclin-dependent kinase 5

Inhibitor-1, the first identified endogenous inhibitor of protein phosphatase 1 (PP-1), was previously reported to be a substrate for cyclin-dependent kinase 5 (Cdk5) at Ser⁶⁷. Further investigation has revealed the presence of an additional Cdk5 site identified by mass spectrometry and confirmed by site-directed mutagenesis as Ser⁶. Basal levels of phospho-Ser⁶ inhibitor-1, as detected by a phosphorylation state-specific antibody against the site, existed in specific regions of the brain and varied with age. In the striatum, basal in vivo phosphorylation and dephosphorylation of Ser⁶ were mediated by Cdk5, PP-2A, and PP-1, respectively. Additionally, calcineurin contributed to dephosphorylation under conditions of high Ca²⁺. In biochemical assays the function of Cdk5-dependent phosphorylation of inhibitor-1 at Ser⁶ and Ser ⁶⁷ was demonstrated to be an intramolecular impairment of the ability of inhibitor-1 to be dephosphorylated at Thr³⁵; this effect was recapitulated in two systems in vivo. Dephosphorylation of inhibitor-1 at Thr³⁵ is equivalent to inactivation of the protein, as inhibitor-1 only serves as an inhibitor of PP-1 when phosphorylated by cAMP-dependent kinase (PKA) at Thr³⁵. Thus, inhibitor-1 serves as a critical junction between kinase- and phosphatase-signaling pathways, linking PP-1 to not only PKA and calcineurin but also Cdk5.