A constraint network of interactions: protein–protein interaction analysis of the yeast type II phosphatase Ptc1p and its adaptor protein Nbp2p

Abstract

IntroductionInactivation of proteins that participate in more than one cellular process leads to a variety of apparently unconnected phenotypes. Understanding the molecular cause for each phenotype might reveal how seemingly independent cellular processes are regulated and coordinated in the cell. Genome-wide gene interaction data based on the simultaneous inactivation of more than one gene greatly facilitate this inherently complex analysis because genes with pleiotropic phenotypes often occupy central positions in the corresponding interaction networks (Costanzo et al., 2010;Tong et al., 2004). By assigning physical connections, protein-protein interaction maps provide the necessary complementary information. Interpretation of these maps is usually not straightforward. Genetic interactions can result from complex functional relationships between the investigated pairs of genes and protein interaction maps are generally projections of contacts that occur at different times and places in the cell. To transform protein interaction data into mechanistically meaningful models, it is necessary to resolve these projections into their different interaction planes. We define an interaction plane or state as the sum of all simultaneously occurring contacts. Ideally, these states should be defined by time-and space-resolved in vivo studies. However, these studies are technically demanding and usually not suited for measuring multiple contacts (Maeder et al., 2007). Using the protein pair Ptc1p-Nbp2p of the yeast Saccharomyces cerevisiae as an example and the split-ubiquitin method (SplitUb) as the experimental tool, we present an alternative approach for defining interaction states. The derived constraint interaction network reduces the number of possible states and thus provides a useful framework for model building and the initiation of more detailed studies.

Wide-Ranging Effects of the Yeast Ptc1 Protein Phosphatase Acting Through the MAPK Kinase Mkk1

“…Remarkably, only the mutation of Mkk1, one of the MAPKKs of the CWI pathway, was able to substantially suppress all the phenotypes of the ptc1 mutant tested in the screen (Figure 1 and Figure 2), as well as additional defects attributable to the ptc1 mutation (Figure 3 and Figure 4). This finding reinforces previous evidence linking Ptc1 to the CWI pathway (Huang and Symington 1995;Gonzalez et al 2006;Hruby et al 2011;Stanger et al 2012;SacristanReviriego et al 2015) and strongly suggests that abnormal activation of the CWI pathway is at the basis of many of the defects derived from the absence of Ptc1. It must be noted that an excessive activation of the CWI pathway can be deleterious, as observed when hyperactive alleles of Pkc1 or Mkk1 are overexpressed (Watanabe et al 1995;Martin et al 2000).…”

“…As shown in Figure 5B, overexpression of Ptc1 was able to reduce the strong Slt2 phosphorylation promoted by Bck1 CT . Because this version does not contain the N-terminal Nbp2-binding proline-rich motif (Hruby et al 2011), we previously confirmed that Nbp2 is not necessary for overexpressed Ptc1 to act on the CWI pathway ( Figure S2). Bck1 CT also lacks the phosphorylation sites necessary for its activation by Pkc1 (Levin et al 1994).…”

“…Cells lacking Ptc1 display higher than normal amounts of active Slt2 (Du et al 2006;Gonzalez et al 2006) and show increased expression of diverse genes (MLP1, CRH1, and SED1) regulated by the Slt2 MAPK module (Gonzalez et al 2006). More recently, Nbp2 has been described to mediate the interaction between Ptc1 and different kinases, including Bck1 (Hruby et al 2011;Stanger et al 2012). In agreement, elimination of Nbp2 results in increased sensitivity to cell-wall stressors.…”

Septin rings act as template for myosin higher-order structures and inhibit redundant polarity establishment

“…3A,B) (Hruby et al, 2011;Labedzka et al, 2012). Deleting 229 or 529 residues from the C-terminus of Myo1p (Myo1 1-1700 , Myo1 1-1400 ) hardly affected the interaction with Bni5p whereas a Myo1-fragment covering only the N-terminal 1100 residues displayed only very weak binding (Fig.…”

Ptc1 Protein Phosphatase 2C Contributes to Glucose Regulation of SNF1/AMP-activated Protein Kinase (AMPK) in Saccharomyces cerevisiae

“…Analysis of the ptc1⌬ mutant using DNA microarrays showed no significant effects on expression of glucoseregulated genes (21,22). Ptc1 binds to the adaptor protein Nbp2, which mediates its association with multiple protein kinases, but SNF1 was not identified as an interaction partner (23). We here examine the effects of the ptc1⌬ mutation and Ptc1 overexpression, in combination with reg1⌬ and/or sit4⌬ mutations, on Thr-210 phosphorylation of wild-type and mutant forms of SNF1.…”