Hr202 and Tyr204 in its activation loop, web sites which are dephosphorylated by many different phosphatases inside certain cellular contexts(Patterson et al. 2009, Paul et al. 2003, Piserchio et al. 2012a) (Li et al. 2013). Each in corticostriatal culture and in vivo, STEP regulates neuronal activities mainly by targeting temporal ERK activation-loop phosphorylation (Paul et al. 2003, Valjent et al. 2005, Venkitaramani et al. 2009). Even though cellular research have Dopamine β-hydroxylase supplier detected the interaction of ERK with STEP (Munoz et al. 2003), direct quantitative measurement of phospho-ERK dephosphorylation by STEP in vitro with purified proteins has not been reported. To start to know the molecular mechanism of phospho-ERK dephosphorylation by STEP, we prepared double-phosphorylated ERK and several protein phosphatases at high purity to compare the activities of various phosphatases toward phospho-ERK (Fig 1A and 1B). As opposed to STEP, the Ser/Thr phosphatase PPM1A selectively dephosphorylates pT202 ofJ Neurochem. Author manuscript; available in PMC 2015 January 01.Li et al.PageERK both in vivo and in vitro (Zhou et al. 2002, Li et al. 2013); in contrast, two other tyrosine phosphatases, BDP-1 and PTP-MEG2, have not been directly linked to phosphoERK dephosphorylation. Applying these phosphatases as controls, we investigated whether or not STEP is an efficient and tyrosine-specific ERK phosphatase in vitro. We first examined ERK dephosphorylation by distinct phosphatases using a distinct antibody that recognises ERK activation-loop phosphorylation (pT202EpY204). In comparison with PTP-MEG2 and BDP1, both STEP and PPM1A displayed efficient catalytic activity toward dual-phosphorylated ERK with equimolar phosphatase inputs (Fig 1). To examine regardless of whether STEP specifically dephosphorylated pY204 rather than pT202, we next monitored dephosphorylation on residue pY204 employing the specific phospho-tyrosine antibody pY350. Although STEP removed many of the phospho-tyrosine on double-phosphorylated ERK, PPM1A showed tiny effect on pY204 (Fig 1A and D). This outcome confirmed that STEP hydrolysed pY204, but didn’t exclude the possibility that STEP dephosphorylated pT202. Therefore, we subsequent monitored the time course of ERK2-pT202pY204 dephosphorylation by CB2 drug sequentially adding STEP and PPM1A. As soon as reaction reached plateau, STEP remedy only lead to 1 equivalent of inorganic phosphate release, compared to input ERK protein. Subsequent inputting PPM1A resulted in another equivalent of inorganic phosphate release (Fig 1E). The PPM1A was a Ser/Thr specific phosphatse. As a result, PPM1A treated curve reflected dephosphorylation of pT202, and STEP treated curve corresponded to dephosphorylation of pY204. Taken with each other, these benefits demonstrate that STEP is an effective ERK phosphatase that selectively recognises pY204 in vitro, whereas PPM1A is definitely an ERK pT202-specific phosphatase. Kinetic parameters of dephosphorylation of phospho-ERK by STEP The above final results demonstrated that STEP effectively dephosphorylates doublephosphorylated ERK on pY204 in vitro. Nevertheless, the kinetic constant with the enzyme is tricky to identify by western blotting. As a result, to measure the kcat and Km of STEP in ERK dephosphorylation accurately, we utilised a previously established continuous spectrophotometric enzyme-coupled assay to characterise the reaction (Zheng et al. 2012, Zhou et al. 2002). Fig 2A displays the progressive curve of STEP-catalysed ERK dephosphorylation at quite a few diverse phospho-ERK con.
