N identified and characterised; STEP46 and STEP61 are the two key isoforms with phosphatase activities (Sharma et al. 1995). The expression of each STEP46 and STEP61 is enriched in medium spiny neurons on the striatum, but their cellular localisations are diverse: STEP46 is primarily localised to the cytosol, whereas STEP61 has an further 172 residues at its N-terminus that localise it to post-synaptic densities and endoplasmic reticulum (Baum et al. 2010). As a member on the PTP superfamily, STEP participates in neuronal activities by regulating the phosphorylation states of essential components of synaptic plasticity, such as subunits of NMDAR and AMPAR and such kinases as Fyn, p38, and Pyks (Zhang et al. 2008, Xu et al. 2012, Baum et al. 2010). In distinct, STEP negatively regulates the activation of ERK, that is the central hub on the phosphorylation networks that respond to extracellular stimulation. In neuronal cells, ERK activation plays crucial roles in spine stabilisation and transmitting action potentials. Accordingly, enhanced STEP activity accompanied by impaired ERK function has been implicated in neuronal degenerative illnesses. Furthermore,J Neurochem. Author manuscript; EGFR/ErbB1/HER1 Storage & Stability available in PMC 2015 January 01.Li et al.PageSTEP-knockout mice show elevated ERK activation (Venkitaramani et al. 2009) and enhanced hippocampal finding out and memory (Venkitaramani et al. 2011). All these outcomes indicate that especially inhibiting STEP activity toward phospho-ERK has therapeutic prospective in neuronal degenerative illnesses. A damaging regulation of STEP activity could be achieved by establishing specific STEP inhibitors that target the phosphatase active website or by disrupting the interactions of STEP with its substrates. On the other hand, the underlying catalytic mechanisms of STEP towards its substrates remain unknown. Within this study, we aimed to figure out the molecular mechanism of STEP within the dephosphorylation of phospho-ERK, the essential substrate of STEP for neuronal activity modulation, employing combined molecular and enzymologic approaches. Our final results reveal the contributions of important components in mediating distinct ERK-STEP recognition and identify peptide sequence selectivity inside the STEP active site, findings which will help in discovering new STEP substrates and creating distinct approaches to inhibit phospho-ERK dephosphorylation by STEP, potentially curing some neuronal diseases.NIH-PA Author ManuscriptMaterialsMaterial and MethodsPara-nitrophenyl phosphate (pNPP) was obtained from Bio Basic Inc. The Tyr(P)-containing peptides were synthesised and HPLC-purified by China Peptides Co. The Ni2+-NTA resin and HiTrap Q FF column applied in protein purification had been bought from Bio Fundamental Inc. and GE Healthcare, respectively. The phospho-specific anti-ERK1/2-pT202/pY204 Oxazolidinone Formulation Antibody was obtained from Cell Signaling, the anti-flag M2 antibody was bought from Sigma, the antibody the -Actin Antibody (C4) and the phospho-tyrosine pY-350 antibody was obtained from Santa Cruz Biotechnology. The totally sequenced human PTPN5 cDNA was bought from Thermo Scientific. The expression plasmid for the STEP catalytic domain (STEP-CD) was a generous gift from Dr. Knapp at target discovery institute, U.K., plus the plasmids expressing ERK2 and MEK1 made use of inside the preparation of phospho-ERK have been generous gifts from Dr. Lefkowitz at Duke University, U.S.A. The nerve development element (NGF) was bought from Sino Biological Inc. Cell Culture and Immunoblotting PC12 cells.
