NIPP1-mediated Interaction of Protein Phosphatase-1 with CDC5L, a Regulator of Pre-mRNA Splicing and Mitotic Entry

Abstract

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...

Control of alternative pre-mRNA splicing by Ca++ signals

“…Phosphatases, including PP1 [173][174][175][176][177][178][179][180], PP2C-gamma [181,182], PP2A [178,183], and the pol II CTD phosphatase FCP1 [184], also interact with splicing factors or are known to control splicing. Of these, PP2A binds CaMK IV in a signalling complex [185,186].…”

De Novo Ceramide Regulates the Alternative Splicing of Caspase 9 and Bcl-x in A549 Lung Adenocarcinoma Cells

“…Using an RT-PCR-based assay (Fig. 1, A and B), it was found that overnight treatment of A549 lung adenocarcinoma cells with 20 M D-e-C 6 ceramide (sub-IC 50 dose for a 24-h period in A549 cells) (IC 50 ϭ 37 M) resulted in altering the ratio of the splice variants of Bcl-x and caspase 9 ( Fig. 2, A and B), but not Bax or caspase 2 (data not shown).…”

“…Endogenous ceramide has recently been found to modulate the phosphorylation status of SR proteins in a PP1-dependent manner (49). Several reports have also demonstrated a role for PP1 in regulating alternative splicing, and two spliceosomal targeting subunits for PP1 have been described (38,48,50,51). Therefore, PP1 may play a role in regulating RNA processing in response to stimuli, in particular, it may define a pathway linking ceramide to the regulation of the alternative splicing of apoptosis regulators.…”

Identification of Two RNA cis-Elements That Function to Regulate the 5′ Splice Site Selection of Bcl-x Pre-mRNA in Response to Ceramide

“…Furthermore, endogenous ceramide has been found recently to modulate the phosphorylation status of SR proteins in a PP1-dependent manner (26). PP1 also associates with the splice factor, PTB-associated splicing factor (PSF), and the PP1 regulatory subunit, nuclear inhibitor of PP1 (NIPP1), has been demonstrated to co-localize with splicing factors (15,25,27,28). Finally, dephosphorylation of SR proteins stimulated by PP1 induces alternative 5Ј splice site selection in adeno pre-mRNA in vitro, demonstrating that pre-mRNA splicing events are regulated in a phosphorylationdependent manner (29).…”