Cadmium is a potent inhibitor of PPM phosphatases and targets the M1 binding site

Abstract

The heavy metal cadmium is a non-degradable pollutant. By screening the effects of a panel of metal ions on the phosphatase activity, we unexpectedly identified cadmium as a potent inhibitor of PPM1A and PPM1G. In contrast, low micromolar concentrations of cadmium did not inhibit PP1 or tyrosine phosphatases. Kinetic studies revealed that cadmium inhibits PPM phosphatases through the M1 metal ion binding site. In particular, the negative charged D441 in PPM1G specific recognized cadmium. Our results suggest that cadmium is likely a potent inhibitor of most PPM family members except for PHLPPs. Furthermore, we demonstrated that cadmium inhibits PPM1A-regulated MAPK signaling and PPM1G-regulated AKT signaling potently in vivo. Cadmium reversed PPM1A-induced cell cycle arrest and cadmium insensitive PPM1A mutant rescued cadmium induced cell death. Taken together, these findings provide a better understanding of the effects of the toxicity of cadmium in the contexts of human physiology and pathology.

Expression of Inorganic Pyrophosphatase (PPA1) Correlates with Poor Prognosis of Epithelial Ovarian Cancer

“…The proteins were electrophoresed by 10% SDS-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride (PVDF) membrane, followed by incubation with 5% BSA at room temperature for 1 h. Rabbit anti-human PPA1 antibody (ab96099, Abcam, USA), moue anti-human β-actin antibody (sc-58673, Santa Cruz, CA, USA), rabbit anti-human β-catenin antibody (#9562, Cell Signaling Technology, MA, USA), and moue anti-human p53 antibody (sc-47698, Santa Cruz, CA, USA), were incubated with the PVDF membranes at 4°C overnight, respectively. Secondary antibodies were then added and incubated at room temperature for 1 h, followed by adding electrogenerated chemiluminescent (ECL) and exposed to X-ray films as reported by others (Pan et al 2013). Each experiment was repeated for at least three times.…”

Structure and biosynthesis of fumosorinone, a new protein tyrosine phosphatase 1B inhibitor firstly isolated from the entomogenous fungus Isaria fumosorosea

“…Para-nitrophenyl phosphate (pNPP) was used in enzymatic reactions to determine the intrinsic catalytic activities of PTP1B (Pan et al, 2013). All assays using pNPP as the substrate were performed in a reaction buffer containing 10 mM of NaAc (sodium acetate), 1 mM of ethylene diamine tetraacetic acid, 1 mM of dithiothreitoll at 37°C, pH 5.5.…”

The second-sphere residue T263 is important for the function and catalytic activity of PTP1B via interaction with the WPD-loop

“…The data were collected and analyzed using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA), and the kinetic parameters were obtained by fitting the data to the Michaelis-Menten equation (Eq. (1)) as described previously (Pan et al, 2013).…”

“…All phosphatase assays were performed as previously described at 30 • C (Pan et al, 2013). The phosphatase activities of PTP1B and its mutants were determined using pNPP, the phosphorylated epidermal growth factor receptor peptide acetylDADEpY 992 L and the phosphorylated insulin receptor peptide acetyl-ETDpY 1162 pY 1163 RKGG as substrates.…”

“…The kinetics were monitored by steady-state kinetics using an inorganic phosphate assay as described previously (Pan et al, 2013;Liu et al, 2012). Log(k cat /K m ) and log (k cat ) values were plotted against the leaving-group pK a of the substrates to derive the leaving-group dependence curve.…”