The C-terminus of NIPP1 (nuclear inhibitor of protein phosphatase-1) contains a novel binding site for protein phosphatase-1 that is controlled by tyrosine phosphorylation and RNA binding

Abstract

Nuclear inhibitor of protein phosphatase-1 (NIPP1; 351 residues) is a nuclear RNA-binding protein that also contains in its central domain two contiguous sites of interaction with the catalytic subunit of protein phosphatase-1 (PP1(C)). We show here that mutation of these phosphatase-interaction sites did not completely abolish the ability of NIPP1 to bind and inhibit PP1(C). This could be accounted for by an additional inhibitory phosphatase-binding site in the C-terminal region (residues 311-351), with an inhibitory core corresponding to residues 331-337. Following mutation of all three PP1(C)-binding sites in the central and C-terminal domains, NIPP1 no longer interacted with PP1(C). Remarkably, while both NIPP1 domains inhibited the phosphorylase phosphatase activity of PP1(C) independently, mutation of either domain completely abolished the ability of NIPP1 to inhibit the dephosphorylation of myelin basic protein. The inhibitory potency of the C-terminal site of NIPP1 was decreased by phosphorylation of Tyr-335 and by the addition of RNA. Tyr-335 could be phosphorylated by tyrosine kinase Lyn, but only in the presence of RNA. In conclusion, NIPP1 contains two phosphatase-binding domains that function co-operatively but which are controlled independently. Our data are in agreement with a shared-site model for the interaction of PP1(C) with its regulatory subunits.

Interactor-mediated Nuclear Translocation and Retention of Protein Phosphatase-1

“…Polyhistidine-tagged NIPP1-(143-224) was expressed in bacteria and purified on Ni 2ϩ -Sepharose (24). Bacterially expressed human PP1␥ 1 and PP1␥ 1 -F257A were purified as described by Egloff et al (23) and assayed with glycogen phosphorylase a as the substrate (24).…”

The Protein Phosphatase-1 (PP1) Regulator, Nuclear Inhibitor of PP1 (NIPP1), Interacts with the Polycomb Group Protein, Embryonic Ectoderm Development (EED), and Functions as a Transcriptional Repressor

“…However, this does not necessarily imply that nucleic acids disrupt the NIPP1-PP1 interaction, in particular, because NIPP1 also contains PP1-binding sites outside its central domain (3). Therefore, we have explored whether NIPP1, EED, and PP1 can form a ternary complex in the presence of nucleic acids.…”

“…It is not known how residues 225-329 of NIPP1 contribute to spliceosome assembly. The extreme COOH terminus of NIPP1 (residues 329 -351) does not appear to be involved in spliceosome assembly but has binding sites for PP1 and for A/U-rich single-stranded nucleic acids (3,11). In addition, a synthetic peptide corresponding to residues 329 -351 as well as NIPP1-(225-351), which may exist as a splice variant known as NIPP1␥ (12) or Ard1 (13), displays an endoribonuclease activity with a specificity that is similar to that of the bacterial RNase E (11), a key regulator of the decay and processing of various RNAs.…”

“…NIPP1 contains binding sites for PP1 in its central and COOH-terminal domains (2,3) and is complexed to 30 -50% of the nuclear pool of PP1 (4). The NIPP1-PP1 holoenzyme in nuclear extracts is inactive but can be activated by the phosphorylation of NIPP1 with protein kinase A (Ser-199), protein kinase CK2 (Ser-204), or protein tyrosine kinases of the Src family (Tyr-335) (3,(5)(6)(7). More recently, we demonstrated that NIPP1 is required for a late step in the assembly of spliceosomes (8), which are the complexes of small nuclear RNAs and proteins that catalyze pre-mRNA splicing.…”

“…NIPP1 contains binding sites for PP1 in its central and COOH-terminal domains (2,3) and is complexed to 30 -50% of the nuclear pool of PP1 (4). The NIPP1-PP1 holoenzyme in nuclear extracts is inactive but can be activated by the phosphorylation of NIPP1 with protein kinase A (Ser-199), protein kinase CK2 (Ser-204), or protein tyrosine kinases of the Src family (Tyr-335) (3, 5-7).…”