A Quantitative Chemical Proteomic Strategy for Profiling Phosphoprotein Phosphatases from Yeast to Humans

Abstract

A "tug-of-war" between kinases and phosphatases establishes the phosphorylation states of proteins. While serine and threonine phosphorylation can be catalyzed by more than 400 protein kinases, the majority of serine and threonine dephosphorylation is carried out by seven phosphoprotein phosphatases (PPPs). The PPP family consists of protein phosphatases 1 (PP1), 2A (PP2A), 2B (PP2B), 4 (PP4), 5 (PP5), 6 (PP6), and 7 (PP7). The imbalance in numbers between serine- and threonine-directed kinases and phosphatases led to the early belief that PPPs are unspecific and that kinases are the primary determinants of protein phosphorylation. However, it is now clear that PPPs achieve specificity through association with noncatalytic subunits to form multimeric holoenzymes, which expands the number of functionally distinct signaling entities to several hundred. Although there has been great progress in deciphering signaling by kinases, much less is known about phosphatases.We have developed a chemical proteomic strategy for the systematic interrogation of endogenous PPP catalytic subunits and their interacting proteins, including regulatory and scaffolding subunits (the "PPPome"). PP1, PP2A, PP4, PP5, and PP6 were captured using an immobilized, specific but nonselective PPP inhibitor microcystin-LR (MCLR), followed by protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a single analysis. Here, we combine this approach of phosphatase inhibitor bead profiling and mass spectrometry (PIB-MS) with label-free and tandem mass tag (TMT) quantification to map the PPPome in human cancer cell lines, mouse tissues, and yeast species, through which we identify cell- and tissue-type-specific PPP expression patterns and discover new PPP interacting proteins.

Synergistic Effect of MC-LR and C-Terminal Truncated HBx on HepG2 Cells and Their Effects on PP2A Mediated Downstream Target of MAPK Signaling Pathway

“…The western blot results showed that MC-LR entered HBx 4, HBx 32, and HBx cells in a time-dependent pattern ( Figure 2D). MC-LR is a well-known hepatotoxin that can greatly inhibit intracellular PP2A activity, which regulates HepG2 cell proliferation, differentiation, and invasion as well as the cell cycle (Sun et al, 2014;Lyons et al, 2018). Similarly, ctHBx could also affect the proliferation migration and invasion capabilities of HepG2 cells (Iavarone et al, 2003;Zhang et al, 2008).…”

PPP3CB Inhibits Migration of G401 Cells via Regulating Epithelial-to-Mesenchymal Transition and Promotes G401 Cells Growth

“…Vital hallmarks change in the process of EMT, such as downregulation of epithelial markers (E-cadherin) and the upregulation of mesenchymal marker (Vimentin) [6]. The PPP family has seven members, including Protein Phosphatases1 (PP1), 2A (PP2A), and 2B (PP2B) [7]. These members are involved in EMT through regulating the expression of EMT markers and modulating a broad range of signaling pathways.…”

Cellular toxicity of iHAP1 and DT-061 does not occur through PP2A-B56 targeting

“…1B-C, Table S1). In a complementary approach, we incubated lysates from untreated or iHAP1/DT-061 treated HEK-293T cells with microcystin coupled to beads and analysed by mass spectrometry the composition of endogenous PPP components which were pulled down by microcystin [15] (Fig. 1D, Table S2).…”

“…PIB pull-downs were analysed on a Q-Exactive Plus quadrupole Orbitrap mass spectrometer (ThermoScientific) equipped with an Easy-nLC 1000 (ThermoScientific) and nanospray source (ThermoScientific) as previously described [15]. Raw data were searched using COMET (release version 2014.01) in high resolution mode [46] against a target-decoy (reversed) [47] version of the human proteome sequence database (UniProt; downloaded 2/2020) with a precursor mass tolerance of +/- 1 Da and a fragment ion mass tolerance of 0.02 Da, and requiring fully tryptic peptides (K, R; not preceding P) with up to three mis-cleavages.…”