Neuronal Cdc2-like protein kinase (Cdk5/p25) is associated with protein phosphatase 1 and phosphorylates inhibitor-2

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 Thr(72) by glycogen synthase kinase 3 (GSK3). We have found that PP1 LB in bovine brain extract is also activated upon preincubation with ATP/Mg. However, blocking GSK3 action by LiCl inhibited only similar to 29% of PP1 activity and indicated that GSI(3 is not the sole PP1 .I-2 activator in the brain. When bovine brain extract was analyzed by gel filtration PPI .I-2 and neuronal CdcS-like protein kinase (NCLK), a heterodimer of Cdk5 and the regulatory p25 subunit, co-eluted as a similar to 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, PPI 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 PPI I-S in an ATP/Mg-dependent manner. NCLK bound to PP1 through its CdkS subunit and the PP1 binding region was localized to CdkS residues 28-41. Our data demonstrate that in brain extract PP1 .I-2 and NCLK are associated within a complex of similar to 450 kDa and suggest that NCLK is one of the PPI I-8-activating kinases in the mammalian brain.