Inhibitor 1 (I-1) is a protein inhibitor of protein phosphatase 1 (PP1), a major 
eukaryotic Ser/Thr phosphatase. Nonphosphorylated I-1 is inactive, whereas 
phosphorylated I-1 is a potent PP1 inhibitor. I-1 is phosphorylated in vivo on 
Thr(35) and Ser(67). Thr(35) is phosphorylated by cAMP-dependent protein kinase 
(A kinase), and Thr(35)-phosphorylated I-1 inhibits PP1. Until now the kinase 
that phosphorylates Ser(67) had not been identified and the physiological role 
of Ser(67) phosphorylation was unknown. In this study we detected a high level 
of kinase activity in brain extract when a glutathione S-transferase (GST) 
fusion I-1 mutant containing an Ala substituted for Thr(35) [GST-I-1(T35A)] was 
used as the substrate. GST-I-1(T35A) kinase and neuronal cdc2-like protein 
kinase (NCLK) in the brain extract could not be separated from each other by a 
series of sequential chromatographies. GST-I-1(T35A) kinase immunoprecipitated 
with anti-NCLK antibody from kinase-active column fractions. Purified 
NCLK-phosphorylated GST-I-1(T35A) and I-1 (0.7 mole of phosphate per mole of 
I-1). HPLC phosphopeptide mapping, amino acid sequencing, and site-directed 
mutagenesis determined that NCLK phosphorylates Ser(67) of I-1. 
NCLK-phosphorylated I-1 and I-1(T35A) inhibited PP1 with IC(50) values 
approximately 9.5 and 13. 8 nM, respectively. When compared, A 
kinase-phosphorylated I-1 was only approximately 1.2 times more inhibitory than 
NCLK-phosphorylated I-1. Our data indicate that NCLK is a potential in vivo I-1 
kinase and that Thr(35) and Ser(67) phosphorylation independently activate I-1.