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 cyto sol,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 g lycogen synthase kinase 3 (GSK3). We have found that PP1 LB in bovine brain extract is also activated upon preincubation with ATP/Mg. However, blockin g GSK3 action by LiCl inhibited only similar to 29% of PP1 activity and ind icated that GSI(3 is not the sole PP1 .I-2 activator in the brain. When bov ine 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 s ubunit, co-eluted as a similar to 450-kDa size species. The NCLK. from the eluted column fractions bound to PP1-specific microcystin-Sepharose and glu tathione S-transferase (GST)-I-2-coated glutathione-agarose beads. Similarl y, PPI from the eluted column fractions was pulled down with GST-Cdk5-coate d glutathione-agarose beads. In vitro, NCLK( phosphorylated I-2 on Thr72 an d activated PPI I-S in an ATP/Mg-dependent manner. NCLK bound to PP1 throug h its CdkS subunit and the PP1 binding region was localized to CdkS residue s 28-41. Our data demonstrate that in brain extract PP1 .I-2 and NCLK are a ssociated within a complex of similar to 450 kDa and suggest that NCLK is o ne of the PPI I-8-activating kinases in the mammalian brain.