Ser67-phosphorylated inhibitor 1 is a potent protein phosphatase 1 inhibitor.
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.
Huang
K X
KX
Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, and Department of Neurology and Neurosurgery, McGill University, 3755 Cote Ste-Catherine Road, Montreal, QC, H3T 1E2.
Paudel
H K
HK
D019943:Amino Acid Substitution; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002850:Chromatography, Gel; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases / Q000302:isolation & purification / Q000502:physiology*; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000502:physiology*; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D010766:Phosphorylation; D010768:Phosphoserine / Q000737:chemistry*; D054645:Protein Phosphatase 1; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity
D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D010768:Phosphoserine; D005982:Glutathione Transferase; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1