Neuronal Cdc2-like Protein Kinase (Cdk5/p25) Is Associated with Protein Phosphatase 1 and Phosphorylates Inhibitor-2

Abstract

Protein phosphatase 1 (PP1) is complexed with inhibitor 2 (I-2) in the cytosol. In rabbit muscle extract PP1·I-2 is activated upon preincubation with ATP/Mg. This activation is caused by phosphorylation of I-2 on Thr72 by glycogen synthase kinase 3 (GSK3). We have found that PP1·I-2 in bovine brain extract is also activated upon preincubation with ATP/Mg. However, blocking GSK3 action by LiCl inhibited only ∼29% of PP1 activity and indicated that GSK3 is not the sole PP1·I-2 activator in the brain. When bovine brain extract was analyzed by gel filtration PP1·I-2 and neuronal Cdc2-like protein kinase (NCLK), a heterodimer of Cdk5 and the regulatory p25 subunit, co-eluted as a ∼450-kDa size species. The NCLK from the eluted column fractions bound to PP1-specific microcystin-Sepharose and glutathione S-transferase (GST)-I-2-coated glutathione-agarose beads. Similarly, PP1 from the eluted column fractions was pulled down with GST-Cdk5-coated glutathione-agarose beads. In vitro, NCLK phosphorylated I-2 on Thr72 and activated PP1·I-2 in an ATP/Mg-dependent manner. NCLK bound to PP1 through its Cdk5 subunit and the PP1 binding region was localized to Cdk5 residues 28-41. Our data demonstrate that in brain extract PP1·I-2 and NCLK are associated within a complex of ∼450 kDa and suggest that NCLK is one of the PP1·I-2-activating kinases in the mammalian brain.