Protein tyrosine phosphatase, PTP1B, expression and activity inrat corneal endothelial cells

[摘要] Purpose: The current studies were conducted to determine whether theprotein tyrosine phosphatase, PTP1B, plays a role in regulatingepidermal growth factor receptor (EGFR) Tyr992 phosphorylation and cellcycle entry in rat corneal endothelial cells.Methods: Corneas were obtained from male Sprague-Dawley rats. PTP1BmRNA and protein expression were compared in confluent and subconfluentcells by RT-PCR and western blots. Immunocytochemistry was used todetermine the subcellular localization of both PTP1B and EGFR followingepidermal growth factor (EGF) stimulation. Western blots were used toanalyze the time-dependent effect of EGF on phosphorylation of EGFRTyr992 plus or minus CinnGEL 2Me, an inhibitor of PTP1B activity. Theeffect of PTP1B inhibition on cell cycle entry was determined bycalculating the percent of Ki67-positive cells following EGF treatment.Results: PTP1B mRNA expression was similar in confluent andsubconfluent cells, but PTP1B protein was expressed at 3 fold higherlevels in subconfluent cells. Positive staining for PTP1B was localizedin vesicular structures below the plasma membrane. EGFR staining waslocated at cell-cell borders in untreated endothelium, but was mainlycytoplasmic by 15 min after EGF treatment. In control cultures,phosphorylation of EGFR Tyr992 peaked by 5 min following EGF stimulationand rapidly decreased to basal levels by 30 min. In cultures pretreatedwith CinnGEL 2Me, Tyr992 phosphorylation peaked 2 min following EGFaddition and was consistently sustained at a higher level than controlsuntil 60 min after treatment. By 18 h following EGF treatment, culturespretreated with CinnGEL 2Me exhibited a 1.7 fold increase in the numberof Ki67-positive cells compared with control cultures.Conclusions: Comparison of PTP1B mRNA and protein levels indicatesthat PTP1B expression is regulated mainly at the protein level and ishigher in subconfluent cells. PTP1B was located in vesicles below theplasma membrane. The fact that EGFR is internalized in response to EGFstimulation suggests that it could interact with and be regulated byPTP1B. The ability of PTP1B inhibitor to sustain EGFR Tyr992phosphorylation and increase the number of Ki67-positive cells indicatesthat PTP1B plays a role in the negative regulation of EGF-inducedsignaling and helps suppress cell cycle entry.

[学科分类] 生物化学/生物物理