Phosphorylation of protein phosphatase inhibitor-1 by protein kinase C

Inhibitor-1 becomes a potent inhibitor of protein phosphatase 1 when phosphorylated by cAMP-dependent protein kinase at Thr³⁵. Moreover, Ser⁶⁷ of inhibitor-1 serves as a substrate for cyclin-dependent kinase 5 in the brain. Here, we report that dephosphoinhibitor-1 but not phospho-Ser⁶⁷ inhibitor-1 was efficiently phosphorylated by protein kinase C at Ser⁶⁵ in vitro. In contrast, Ser⁶⁷ phosphorylation by cyclin-dependent kinase 5 was unaffected by phospho-Ser ⁶⁵. Protein kinase C activation in striatal tissue resulted in the concomitant phosphorylation of inhibitor-1 at Ser⁶⁵ and Ser ⁶⁷, but not Ser⁶⁵ alone. Selective pharmacological inhibition of protein phosphatase activity suggested that phospho-Ser ⁶⁵ inhibitor-1 is dephosphorylated by protein phosphatase 1 in the striatum. In vitro studies confirmed these findings and suggested that phospho-Ser⁶⁷ protects phospho-Ser⁶⁵ inhibitor-1 from dephosphorylation by protein phosphatase 1 in vivo. Activation of group I metabotropic glutamate receptors resulted in the up-regulation of diphospho-Ser⁶⁵/Ser⁶⁷ inhibitor-1 in this tissue. In contrast, the activation of N-methyl-D-aspartate-type ionotropic glutamate receptors opposed increases in striatal diphospho-Ser⁶⁵/Ser ⁶⁷ inhibitor-1 levels. Phosphomimetic mutation of Ser⁶⁵ and/or Ser⁶⁷ did not convert inhibitor-1 into a protein phosphatase 1 inhibitor. On the other hand, in vitro and in vivo studies suggested that diphospho-Ser⁶⁵/Ser⁶⁷ inhibitor-1 is a poor substrate for cAMP-dependent protein kinase. These observations extend earlier studies regarding the function of phospho-Ser⁶⁷ and underscore the possibility that phosphorylation in this region of inhibitor-1 by multiple protein kinases may serve as an integrative signaling mechanism that governs the responsiveness of inhibitor-1 to cAMP-dependent protein kinase activation.