Binding of the Concave Surface of the Sds22 Superhelix to the α4/α5/α6-Triangle of Protein Phosphatase-1

Abstract

Functional studies of the protein phosphatase-1 (PP1) regulator Sds22 suggest that it is indirectly and/or directly involved in one of the most ancient functions of PP1, i.e. reversing phosphorylation by the Aurora-related protein kinases. We predict that the conserved portion of Sds22 folds into a curved superhelix and demonstrate that mutation to alanine of any of eight residues (Asp 148 Among the protein phosphatases that occur in all studied eukaryotic lineages, the Ser/Thr-specific protein phosphatases of type-1 are the best conserved, with more than 70% of their residues nearly invariant (1). This conservation extends well beyond structurally and catalytically important residues to include exposed residues involved in the binding of regulatory proteins. As a catalytic subunit, PP1 1 depends on the interaction with one or two regulatory subunits for subcellular localization, substrate specificity, and activity regulation (1, 2). Eukaryotic cells contain a large variety of regulatory subunits of PP1, which account for the diversified action of this phosphatase. We have recently proposed that PP1 acquired an essential function during early eukaryotic evolution by the development of sites for interaction with a primordial regulatory subunit(s) (1). This essential primordial function and the sequential acquirement of additional interaction sites and functions would then have impeded further mutation of the corresponding portion(s) of the surface. The phylogenetic distribution of PP1 indicates that this primordial function must have been acquired before the divergence of the extant eukaryotic lineages. One of the most ancient functions of PP1 is to dephosphorylate substrates of the Aurora-related protein kinases, and this is essential for the completion of mitosis (3). The regulatory subunit(s) associated with this function of PP1 remain unknown, but the protein Sds22 (38 kDa) has emerged as a prime candidate. First, both yeast and mammalian Sds22 have been shown to interact with PP1 and to be part of a complex with PP1 that is enriched in the nucleus (4 -7). Second, the Sds22 encoding gene was identified independently in fission and in budding yeast as an extra-copy suppressor of the temperaturesensitive mitotic arrest phenotypes that are associated with certain mutations of PP1 (4,5,8). Deletion of the Sds22-encoding gene caused a similar mitotic arrest, and this phenotype could be complemented by the overexpression of PP1 (4,5,8). Third, the conditionally lethal phenotype in budding yeast that was conferred by a loss-of-function mutation of the Aurora-related kinase Ipl1 (Ipl1-2), was largely relieved by the expression of certain temperature-sensitive mutant versions of Sds22 or PP1 (9, 10). The mutant Sds22 version that rescued the Ipl1-2 phenotype showed a decreased ability to interact with PP1. The expression of this mutant Sds22 did not affect the cellular levels of PP1 or Sds22, but drastically reduced the nuclear level of PP1 and caused a redistribution of the nuclear pool of PP1 (9).Hitherto, little ...

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

“…The immunoprecipitation of EGFP-PP1␥ 1 -⌬286 -323 and EGFP-PP1␥ 1 -F257A did not result in the co-immunoprecipitation of two RVXF-containing interactors (NIPP1 and PNUTS) but showed an increased co-immunoprecipitation of Sds22, which does not contain a functional RVXF motif. The increased interaction of EGFP-PP1␥ 1 -⌬286 -323 with Sds22 has been reported previously and has been explained by a lack of competition with RVXF-containing interactors of PP1 (26). Our observation that the cytoplasmically retained mutants of EGFP-PP1␥ 1 show an increased interaction with Sds22 is initial evidence that the nuclear targeting of EGFP-PP1␥ 1 is not mediated by Sds22.…”

“…PP1␥ 1 -F257A could not be re-targeted to the nucleus by the overexpression of Sds22 (Fig. 5), which interacts with a fragment of PP1 that is remote from the RVXF-binding channel (26). This was an unexpected finding, because FLAG-Sds22 is …”

“…We therefore generated two PP1␥ 1 variants that are mutated in the RVXF-binding channel, which is involved in the binding of most interactors of PP1 (see Introduction). EGFP-PP1␥ 1 -⌬286 -323, which is truncated just before the last ␤-strand (␤14) that lines the RVXF-binding channel (26), was largely cytoplasmic and sometimes showed a speckled distribution in the cytoplasm (Fig. 1C).…”

Nuclear Targeting of Protein Phosphatase-1 by HIV-1 Tat Protein

“…Therefore the entire Tat-(49 -59) sequence appears to interact with PP1. Taken together, our results indicate that two sequences of Tat, 29 KCCFHCQVCFITKAL 43 and 49 RKKRRQR-RRAH 59 , are likely to contain PP1-interacting amino acids (shown in Fig. 3B).…”

“…The sequences of the DNA constructs were verified by sequencing using a commercial service from Macrogen (Korea). Expression vectors for PP1␥-EGFP and PP1␥-D64N-EGFP are described (29).…”