The Study On Histidine Protein Tyrosine Phosphatase And The Marker Of Cell Autophagy

Protein phosphatases play important functions in cell growth, division and differentiation. They counteract the activities of protein kinase thereby controlling protein phosphorylation and cell signaling pathways. Among protein phosphatases, there are 107 protein tyrosine phosphatases. As crucial regulators of signal transduction, dysfunction of these enzymes causes many human diseases.STS-1 and STS-2 form a sub-family of protein tyrosine phosphatase. STS-1 was initially identified as a suppressor of the T cell receptor signaling pathway. STS-1 and STS-2 have multi-functional structures consisting of UBA domain, SH3 domain, PGM domain, and dimmerization domain. The UBA domain is involved in protein ubiquitination and regulates degradation of STS1 and STS2. The SH3 domain initiates interactions with proline-rich segments of signaling proteins. The dimmerization domain is responsible for formation of STS-1 and STS-2 dimmers. PGM domain is homologous to phosphataseglycerol mutase which catalyzes conversion of 2-phosphoglycerol to 3-phosphoglycerol. Recently, the PGM domain of STS-1 was found to have protein tyrosine phosphatase activity. It dephosphorylates para-nitrophenolphosphate (p-NPP) in biochemical assays and reduces tyrosine phosphorylation of the epidermal growth factor receptor in transfected cells. However, if the PGM domain of STS-2 has similar activity is not known. Based on the high sequence homology of STS-1 and STS-2, we hypothesize that STS-2 is also a protein phosphatase.To confirm our hypothesis, we expressed three fragments containing the PGM domains of STS-1 and STS-2 as non-fusion proteins as well as GST fusion proteins in E. coli cells. These proteins were purified to near homogeneity by using chromatographic procedures. We first analyzed the phosphatase activities of purified non-fusion proteins by using pNPP as a substrate under various conditions. In contrast to STS-1 which displays optimal activity at neutral pH, STS-2 strongly prefer acidic pH. Furthermore, while increase in ionic strength reduces activity of STS-2, this has no effects on STS-1. Overall, STS-1 is more active with a kcat value of 20/second versus 0.1/second for STS-2. We then compared with activity obtained with GST-fusion proteins. Interestingly, while fusion of STS-1 with GST increases its phosphatase activity, GST-STS-2 is much less active then STS-2 alone. Since GST intends to induce protein dimmer formation, the data suggest that dimmerization distinctly regulates activity of STS-1 and STS2.We further analyzed the activity of STS-1 and STS-2 with a recombinant protein substrate designated GST-JAKS and extracts of Jurkat cells which were stimulated with pervanadate to stimulate tyrosine phosphorylation. Both STS-1 and STS-2 effectively dephosphorylated tyrosine phosphorylated proteins with optimal pH 7 for the former and pH4 for the latter. Interestingly, they appeared to selectively dephosphorylate proteins in the Jurkat cell extracts. This indicates different substrate specificity.Finally, we investigated the interaction of STS-1 and STS-2 by analyzing STS-1 activity in the presence of STS-2. The data demonstrated that STS-2 significantly inhibits STS-1. This represents a novel mechanism for the regulation of these enzymes. Together, our study demonstrated that STS-2 is a protein tyrosine phosphatase with distinct feature from STS-1. STS-1 and STS2 may have different biological functions.Autophagy is a catabolic process which causes degradation of a cell's own components through the lysosomal machinery. It plays an important role in cell growth and development. There are different methods for detection of autophagy. In this study, we cloned a chimeric protein containing the mouse microtubule-associated protein light chain 3 beta (LC3b) and enhanced green fluorescent protein (EGFP). When expressed in mammalian cells, the EGFP-mLC3b chimeric protein is diffused in the entire cytosol. Upon treatment of the transfected cells with reagents which induce autophagy, EGFP-mLC3b is localized to punctate intracellular structures resembling autophagosomes. Therefore, EGFP-mLC3b can serve as a marker of autophagy. Our study thus produced a valuable tool for further study of autophagy.