TI - Phosphorylation of protein phosphatase inhibitor -1 by Cdk5 . AB - Protein phosphatase inhibitor-1 is a prototypical mediator of cross-talk between protein kinases and protein phosphatases . Activation of cAMP -dependent protein kinase results in phosphorylation of inhibitor-1 at Thr-35 , converting it into a potent inhibitor of protein phosphatase-1 . Here we report that inhibitor-1 is phosphorylated in vitro at Ser-67 by the proline -directed kinases , Cdk1 , Cdk5 , and mitogen -activated protein kinase . By using phosphorylation state-specific antibodies and selective protein kinase inhibitors , Cdk5 was found to be the only kinase that phosphorylates inhibitor-1 at Ser-67 in intact striatal brain tissue . In vitro and in vivo studies indicated that phospho-Ser-67 inhibitor-1 was dephosphorylated by protein phosphatases-2A and -2B . The state of phosphorylation of inhibitor-1 at Ser-67 was dynamically regulated in striatal tissue by glutamate -dependent regulation of N-methyl-d-aspartic acid -type channels . Phosphorylation of Ser-67 did not convert inhibitor-1 into an inhibitor of protein phosphatase-1 . However , inhibitor-1 phosphorylated at Ser-67 was a less efficient substrate for cAMP -dependent protein kinase . These results demonstrate regulation of a Cdk5-dependent phosphorylation site in inhibitor-1 and suggest a role for this site in modulating the amplitude of signal transduction events that involve cAMP -dependent protein kinase activation .