A Complex of Catalytically Inactive Protein Phosphatase-1 Sandwiched between Sds22 and Inhibitor-3

Abstract

Protein Ser/Thr phosphatase-1 (PP1) associates with a host of proteins to form substrate-specific holoenzymes. Sds22 and Inhibitor-3 (I3) are two independently described ancient interactors of PP1. We show here by various approaches that Sds22 and I3 form a heterotrimeric complex with PP1, both in cell lysates and after purification. The stability of the complex depended on functional PP1 interaction sites in Sds22 and I3, indicating that PP1 is sandwiched between Sds22 and I3. Intriguingly, I3 could not be replaced in this complex by another PP1 interactor with the same PP1 binding motif. In vitro, Sds22 and I3 were potent inhibitors of PP1, but with only some substrates. The inhibition by Sds22 could be reproduced with synthetic Sds22 fragments comprising leucine-rich repeats (LRR) 2 and 5. Sds22 and LRR5 also slowly converted PP1 into a conformation that was inactive with all tested substrates. Cell lysates that were prepared under conditions that prevented the Sds22-induced inactivation of PP1 contained a catalytically inactive complex of Sds22, PP1, and I3, indicating that this complex exists in vivo. Therefore, our studies show that a pool of PP1 is complexly controlled by both Sds22 and I3.

Plasmodium falciparum Inhibitor-3 Homolog Increases Protein Phosphatase Type 1 Activity and Is Essential for Parasitic Survival

“…Plasmids pCAM-HA, pCAM-GFP, and pCAM were kind gifts from Dr. C. Doerig (Inserm-EPFL Joint Laboratory, Switzerland). GST-Ypi1 and GST-I3 recombinant proteins were prepared as described previously (25,26) with pWS93 to tag proteins with HA 3 epitopes, and pBTM116 vectors for yeast two-hybrid experiments have been previously described (27,28). Plasmid construction of pWS93-Ypi1 has been described previously (25), and pBTM116-Glc7 was generated by subcloning the BamHI fragment obtained by digestion of pGAD-Glc7 plasmid reported previously (29).…”

Role of Protein Phosphatase 1 in Dephosphorylation of Ebola Virus VP30 Protein and Its Targeting for the Inhibition of Viral Transcription

“…Because PP1 functions as a heterodimer, we analyzed PP1 distribution in the cells by quantifying expression of its cytoplasmic and nuclear regulatory subunits. Specifically, we analyzed the expression of SIPP1, a nucleocytoplasmic shuttling subunit of PP1 that accumulates in the cytoplasm in UV or X-irradiated cells (14); PNUTS, a major nuclear subunit of PP1 (22); and Sds22, a subunit of PP1 that shuttles PP1 to the nucleus (23). SIPP1 mRNA expression was significantly increased in PP1 KD cells, whereas the expressions of Sds22 and PNUTS mRNAs were not altered (Fig.…”

Assembly and quality control of protein phosphatase 1 holoenzyme involve Cdc48-Shp1 chaperone

“…Progressive depletion of Ypi1 leads to a reduction of nuclear Glc7 and Sds22 proteins and mitotic arrest (Bharucha et al, 2008;Pedelini et al, 2007). A potential homolog of Ypi1 in mammals is known as Inhibitor-3 (also known as PPP1R11), which forms a heterotrimeric complex with Sds22 and the catalytic subunit of PP1 (Lesage et al, 2007). However, unlike yeast Sds22 and Ypi1, mammalian Sds22 and Inhibitor-3 have been shown to be substrate-dependent inhibitors of PP1 in vitro (Lesage et al, 2007).…”