Properties and phosphorylation sites of baculovirus-expressed nuclear inhibitor of protein phosphatase-1 (NIPP-1).
NIPP-1 is the RNA-binding subunit of a major species of protein phosphatase-1 in the nucleus. We have expressed nuclear inhibitor of protein phosphatase-1 (NIPP-1) in Sf9 cells, using the baculovirus-expression system. The purified recombinant protein was a potent (Ki = 9.9 +/- 0.3 pM) and specific inhibitor of protein phosphatase-1 and was stoichiometrically phosphorylated by protein kinases A and CK2. At physiological ionic strength, phosphorylation by these protein kinases drastically decreased the inhibitory potency of free NIPP-1. Phosphorylation of NIPP-1 in a heterodimeric complex with the catalytic subunit of protein phosphatase-1 resulted in an activation of the holoenzyme without a release of NIPP-1. Sequencing and phosphoamino acid analysis of tryptic phosphopeptides enabled us to identify Ser178 and Ser199 as the phosphorylation sites of protein kinase A, whereas Thr161 and Ser204 were phosphorylated by protein kinase CK2. These residues all conform to consensus recognition sites for phosphorylation by protein kinases A or CK2 and are clustered near a RVXF sequence that has been identified as a motif that interacts with the catalytic subunit of protein phosphatase-1.
Vulsteke
V
V
Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.
Beullens
M
M
Waelkens
E
E
Stalmans
W
W
Bollen
M
M
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins; D047390:Casein Kinase II; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D018411:Spodoptera
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D016601:RNA-Binding Proteins; C021042:protein phosphatase inhibitor-1; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1