Role And Molecular Mechanism Of Metal Ions To The Serine/Threonine Phosphatase PP2C And Endothelial Cells

Background and SignificanceProtein phosphatases can be divided into three major families based on their substrate specificity and on the conservation of their catalytic domains. PPMs are a group of phosphatases depend on bivalent cations (Mg2+or Mn2+) for their catalytic activity and are insensitive to the broad-spectrum phosphatase-inhibitor okadaic acid. In humans16different PP2C family members have been identified, which encode for at least22different PP2C isozymes by means of alternative splicing. The functional importance of PP2C isozymes has been shown in a lot of signalling networks controlling cell differentiation, proliferation, growth, survival and metabolism. PP2Ca is expressed in virtually all tissues and participates in the regulation of several important signaling pathways, such as cellular stress signaling. PP2Ca can dephosphorylate and inactivate AMP-activated protein kinase (AMPK), which is the central component of a protein kinase cascade that is activated by hypoxia, heat shock, and metabolic poisoning and so on. AMPK is important for assuring heart tissue homeostasis. PP2Ca is also involved in the regulation of mitogen-activated protein kinase (MAPK) signaling. However, little is known about the regulatory mechanisms of PP2C at the molecular level. Divalent metal ions bind to members of the PPM Ser/Thr phosphatase family and act as either activators or inhibitors. As a prototype of the PPM family, PPM1A was identified to be activated by Mn2+ions but inhibited by Ca2+. A better understanding of cadmium’s function in biological systems, including the interaction between cadmium and phosphatases, is of great importance in treating cadmium exposure-related toxicity.Cadmium has long been recognized as a hazardous material. Human exposure to cadmium can lead to hypertension, cancer, organ dysfunction, and immune system disorders. The half-life of cadmium in the human body is approximately20-30years. The mechanism leading to cadmium toxicity is complicated, and multiple cellular targets have been reported. For example, cadmium can inhibit the functions of zinc finger proteins, regulate intracellular calcium, alter ROS signaling and inhibit DNA repair. In addition, it has been shown that cadmium exposure affects phosphorylation cascades. In cells, protein phosphorylation is precisely regulated by the concerted actions of protein kinases and protein phosphatases. Although long-term exposure to cadmium has been shown to decrease the protein levels of PP5and PP2A-A subunit, it has not been investigated whether cadmium can regulate protein phosphorylation by directly binding to kinases or phosphatases.First PartEffect of metal ions on PP2CObjectivesTo investigate the role of divalent metal ions on PP2C family.Methods1. Plasmid construction and protein expression and purificationThe cDNAs of human PPM1A and PPM1G were subcloned into the PET-30a expression vector with an N-terminal His tag. Expression and purification of pet30a-PPMl A and pet30a-PPM1G were preformed by affinity column with Ni-NTA.2. Study of enzyme kineticsPara-nitrophenyl phosphate (pNPP) was used in enzymatic reactions to determine the intrinsic catalytic activities of PPM1A, PPM1G and their mutants. To determine the kcat and kcat/Km values, the initial rates were measured at various substrate or pseudo-substrate concentrations. Mn2+was used for the majority of the kinetic studies because of its high specific activity and relative stability.ResultsMg2+, Mn2+, Fe2+, Co2+, and Ni2+activated both PPM1A and PPM1G, whereas other metal ions exhibited no detectable or only weak activity. The metal ions with low/no activity might be phosphatase inhibitors and the rankings of the inhibition potencies of these metal ions for the two tested phosphatases were very similar:Cd2+> Zn2+> Hg2+>Ca2+>Sr2+>(Cr2+, Ba2+)>Li+. Cadmium is a potent and competitive inhibitor of PPM1A and PPM1G, with a Ki below1μM, which is approximately1order of magnitude better than the Ki of Zn2+, the second most potent inhibitor in this series of metals. The Mn2+-dependent PPM1A-and PPM1G-catalyzed pNPP hydrolysis reactions inhibited by Cd2+displayed the typical intersecting line pattern for competitive inhibition, with Ki values of300nM and170nM, respectively. Second PartCadmium targets the M1binding site of PPM phosphatasesObjectivesTo investigate the role and mechanism of cadmium on PP2C family.Methods1. Plasmid construction and protein expression and purificationThe cDNAs of human PPM1A and PPM1G were subcloned into the PET-30a expression vector with an N-terminal His tag. To express PPM1A and PPM1G in mammalian cells, human full-length wild-type PPM1A and PPM1G were subcloned into the pcDNA3.1vector containing an N-terminal Flag tag. The16PPM1A mutants and11PPM1G mutants were generated using the QuikChange mutagenesis kit obtained from Stratagene. Expression and purification of pet30a-PPM1A, pet30a-PPM1G and their mutants were preformed by affinity column with Ni-NTA.2. Cadmium inhibition assayThe Ki values for the inhibition of PPMIA, PPMIG and mutants by Cd2+were measured using pNPP as the substrate at varying concentrations of MnCl2.3. Dephosphorylation of Protein Substrates in vitroThe dephosphorylation of a recombinant phosphorylated ERK2protein by PPMIA, D239K or D282A mutant of PPM1A, and PPMIG was tested. The extent of the reaction was analyzed by Western blotting with an anti-ppERK-pT202pY204antibody.4. Functional study1) MAPK signaling and AKT signaling pathways.HEK293cells were transfected with pcDNA3.1-PPMIA, pcDNA3.1-PPM1G or their mutants. At36hours after transfection and after12hours of starvation, the cells were incubated with1.5μM CdCl2for6hours and then stimulated with5ng/ml EGF or0.4M sorbitol. Subsequently, the cells were harvested and lysed. The cell lysates were subjected to SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted with the appropriate antibodies.2) Cell Cycle Assay.HEK293cells were transfected with pcDNA3.1-PPM1A. At36hours after transfection and after12hours of starvation, the cells were incubated with1.5μM CdCl2for24hours. Then, the cell cycle was resumed by adding medium containing10%FBS. The cells were harvested and analyzed using a fluorescence-activated cell sorter.Results1. Cadmium inhibits PPM1A and PPM1G through the M1metal binding site.The D60A and D60N mutations decreased both Mn2+binding and the inhibitory potency of Cd2+by1to2orders of magnitude. The significant impairment of Cd2+-mediated inhibition by both D60A and D60N demonstrates that both the side chain and the negative charge of D60are required for Cd2+coordination. In addition to D60, D239and D282in PPMIA (D441and D496in PPMIG) directly coordinate metal M1. The elimination of the side chains of these aspartic residues resulted in the loss of Cd2+-mediated inhibition by more than one order of magnitude. The inhibitory activities of Cd2+on the D239K mutant of PPM1A and the D441N and D496N mutants of PPM1G were reduced by more than45-fold. The D441N mutation decreased the levels of inhibition by Cd2+, Zn2+, Ca2+and Sr2+by more than10-fold but increased the levels of inhibition by Hg2+, Cr2+and Li+by between2-and7-fold. Therefore, the above results indicate that the unique properties of the M1metal ion binding site contribute to the inhibitory activities of Cd2+. In particular, the negative charge of D441imparts selectivity for coordinating Cd2+, Zn2+, Ca2+and Sr2+but is not required for binding to several other metal ions, such as Mn2+, Hg2+and Cr2+. The inhibitory effect of Cd2+on PPM family phosphatases are mediated through the unique effect of this ion on the M1metal ion binding site, and one of the special cadmium-binding features is the negative charge of the conserved D441residue of PPM1G.2. Cadmium suppresses PPM1A-and PPM1G-regulated signaling pathways in cells at a low micromolar concentration.Overexpression of PPM1A by approximately2-fold relative to the endogenous protein level in HEK293cells significantly blocked sorbitol-induced P38and JNK-1phosphorylation. This result was drastically reversed by the application of a low micromolar concentration of Cd2+. Overexpression of PPM1A resulted in a reduction of ERK phosphorylation at the pT202/pY204site by half. This effect could be reversed by adding1.5μM Cd2+. Overexpression of PPM1G promoted AKT phosphorylation at pT473. When1.5μM Cd2+was added, the effect of PPM1G on AKT phosphorylation was completely blocked. Overexpression of D239K by5-fold relative to endogenous PPM1A still significantly blocked sorbitol-induced P38and JNK-1phosphorylation, but1.5μM Cd2+had much weaker effects on the D239K mutant of PPM1A.3. Cadmium reverses the function of PPMIA on cell cycle and D239K mutant of PPM1A rescued cadmium-induced PC12cell apoptosis.The overexpression of PPM1A in HEK293cells led to the pronounced accumulation of cells in G2/M phase, with an approximately100%increase compared with control cells. The addition of1.5μM Cd2+greatly reversed the increase in the proportion of cells in G2/M. Application of Cd2+induced PC12cells apoptosis. This effect can be partially reversed by overexpression of D239K of PPMIA, a Cd2+insensitive mutant, validating the concept that Cd2+can regulate PPM1A functioning by inhibiting its activity in cells. Third PartEffect of cadmium on endothelial cellsObjectivesTo study the effect of cadmium on endothelial cells.Methods1. Plasmid constructionTo express PPM1A in mammalian cells, human full-length wild-type PPM1A were subcloned into the pcDNA3.1vector containing an N-terminal Flag tag.2. Cell cultureHuman umbilical vein endothelial cells (HUVECs) were cultured in F-12K medium containing O.lmg/ml heparin,0.03-0.05mg/ml endothelial cell growth supplement (ECGS) and10％fetal calf serum (FBS) in.3. MTT assay and TUNEL assay(1) Detect the cell proliferation and apoptosis after the cells were incubated with Cd2+of different concentration or at different times.(2) Detect the cell proliferation and apoptosis after the cells were incubated with1μM Cd2+for24hours followed by overexpression of PPM1A.(3) Detect the cell proliferation after incubated with1μM Cd2+for24hours followed by knockdown of PPM1A.3. Detection of the effect of Cd2+on intracellular GSH and SOD activity.Detect the intracellular GSH and SOD activity after cells were incubated with different concentrations of Cd2+for24hours.ResultsThe effect of Cd2+on HUVECs proliferation and apoptosis is dose-dependent and time-dependent. Cd2+of low concentration will promote cell proliferation, whereas Cd2+of high concentration will promote apoptosis. Moreover, Cd2+of low concentration can inhibit PPMlA-induced apoptosis; Cd2+of high concentration could induce apoptosis by increasing the oxidative stress.. Conclusion1. Mg2+, Mn2+, Fe2+, Co2+and Ni2+can activate PPM1A and PPM1G; Cd2+, Zn2+, Hg2+, Ca2+, Sr2+, Cr2+and Ba2+can inhibit PPM1A and PPM1G.2. Cadmium is a potent inhibitor of PP2C by the M1metal ion binding site.3. In cells, cadmium could inhibit PPM1A-regulated MAPK signaling pathway and PPM1G-regulated AKT signaling pathway.4. Cadmium can inhibit PPM1A-induced cell cycle arrest.5. The effect of Cd2+on HUVECs proliferation and apoptosis is dose-dependent and time-dependent.6. Cd2+of low concentration can inhibit PPM1A-induced apoptosis...