NIPP-1 is a subunit of the major nuclear protein phos-
phatase-1 (PP-1) in mammalian cells and potently inhib-
its PP-1 activity in vitro. Using yeast two-hybrid and
co-sedimentation assays, we mapped a PP-1-binding site
and the inhibition function to the central one-third do-
main of NIPP-1. Full-length NIPP-1 (351 residues) and
the central domain, NIPP-1143–217, were equally potent
PP-1 inhibitors (IC505 0.3 nM). Synthetic peptides span-
ning the central domain of NIPP-1 further narrowed the
PP-1 inhibitory function to residues 191–200. A second,
noninhibitory PP-1-binding site was identified by far-
Western assays with digoxygenin-conjugated catalytic
subunit (PP-1C) and included a consensus RVXF motif
(residues 200 –203) found in many other PP-1-binding
proteins. The substitutions, V201A and/or F203A, in the
RVXF motif, or phosphorylation of Ser199or Ser204,
which are established phosphorylation sites for protein
kinase A and protein kinase CK2, respectively, pre-
vented PP-1C-binding by NIPP-1191–210 in the far-West-
ern assay. NIPP-1191–210 competed for PP-1 inhibition by
full-length NIPP-11–351, inhibitor-1 and inhibitor-2, and
dissociated PP-1Cfrom inhibitor-1- and NIPP-1143–217-
Sepharose but not from full-length NIPP-11–351-Sepha-
rose. Together, these data identified some of the key
elements in the central domain of NIPP-1 that regulate
PP-1 activity and suggested that the flanking sequences
stabilize the association of NIPP-1 with PP-1C.
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