NIPP1 is a regulatory subunit of a species of protein phosphatase-1 (PP1) that co-localizes with splicing factors in nuclear speckles. We report that the N-terminal third of NIPP1 largely consists of a Forkhead-associated (FHA) protein interaction domain, a known phosphopeptide interaction module. A yeast two-hybrid screening revealed an interaction between this domain and a human homolog (CDC5L) of the fission yeast protein cdc5, which is required for G 2 /M progression and pre-mRNA splicing. CDC5L and NIPP1 co-localized in nuclear speckles in COS-1 cells. Furthermore, an interaction between CDC5L, NIPP1, and PP1 in rat liver nuclear extracts could be demonstrated by co-immunoprecipitation and/or co-purification experiments. The binding of the FHA domain of NIPP1 to CDC5L was dependent on the phosphorylation of CDC5L, e.g. by cyclin E-Cdk2. When expressed in COS-1 or HeLa cells, the FHA domain of NIPP1 did not affect the number of cells in the G 2 /M transition. However, the FHA domain blocked ␤-globin pre-mRNA splicing in nuclear extracts. A mutation in the FHA domain that abolished its interaction with CDC5L also canceled its anti-splicing effects. We suggest that NIPP1 either targets CDC5L or an associated protein for dephosphorylation by PP1 or serves as an anchor for both PP1 and CDC5L.Type 1 protein phosphatases (PP1) 1 belong to the PPP family of Ser/Thr protein phosphatases and regulate diverse cellular processes such as transcription, pre-mRNA splicing, intracellular transport, and metabolism (1-3). They consist of a single catalytic subunit (PP1 C ) and one or two regulatory subunits. The regulatory subunits act as substrate specifiers and anchor the holoenzymes in specific cell compartments in close vicinity to their substrates. In addition, the regulatory subunits mediate the control of the holoenzymes by hormones and growth factors through interaction with allosteric effectors or through phosphorylation by specific protein kinases. It has been estimated that mammalian cells contain tens of different regulatory proteins of PP1 (4). Altogether about 20 of these have already been characterized and cloned, including the glycogenbinding G-subunits, the myosin-binding M-subunits, the cytosolic regulator inhibitor-1, and the nuclear RNA-binding protein NIPP1 (1-3). Recent investigations have revealed that these regulatory proteins have multiple points of interaction with PP1 C , including a common phosphatase binding motif with the consensus sequence RVXF (5-10). In addition, most regulatory subunits contain domains that mediate the binding to substrates (e.g. myosin for the M-subunits) and/or a subcellular structure (e.g. glycogen for the G-subunits) to which the substrates are bound.In nuclear extracts, NIPP1 (39 kDa) is present as an inactive complex with PP1 C , termed PP1N NIPP1 (11). This heterodimer can be activated by phosphorylation of up to 4 Ser/Thr residues in the central domain of NIPP1 by protein kinases A and CK2 (12), which disrupts its interaction with PP1 C via the RVXF motif without dis...

The activity of protein phosphatase-1 in rat liver nuclei (PP-1N) was decreased by up to 97% by associated inhibitory polypeptides, depending on the assay and extraction conditions. These inhibitors were rapidly degraded by endogenous proteases, resulting in the accumulation of active heat-stable intermediates. Two major species of PP-1N could be differentiated by fractionation of a nuclear extract. PP-1NR111 contained, besides the delta-isoform of the catalytic subunit, an inhibitory polypeptide of 111 kDa. PP-1NR41 was found to be an inactive heterodimer between the delta-isoform of the catalytic subunit and NIPP-1, a nuclear inhibitor of PP-1, which in its undegraded form is heat labile and migrates during SDS-polyacrylamide gel electrophoresis as a polypeptide of 41 kDa. Native hepatic NIPP-1 displayed a reduced affinity for the catalytic subunit after phosphorylation by protein kinase A in vitro and after glucagon-induced phosphorylation in vivo.

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.