Cloning of Human Lens Low Molecular Weight Protein Tyrosine Phosphatase (LMW–PTP) and its Role in PDGF–induced Signaling in Human Lens Epithelial Cells

Abstract

Abstract: : Purpose: This study is to demonstrate the presence of the low molecular weight protein tyrosine phosphatase (LMW–PTP) in human lens epithelial (HLE) cells and its role in platelet–derived growth factor (PDGF)–induced signaling. Methods: Total PTP activity in both HLE B3 and SRA01/04 cells was analyzed using either pNPP or phosphotyrosine as substrate. LMW–PTP in cell lysate was detected using immunoblot against anti–human LMW–PTP antibody. RT–PCR was applied to amplify LMW–PTP cDNA from total RNA with primers designed upon the sequence of acid phosphatase from human red blood cells. PCR product was purified and cloned into pCR3.1 vector for sequencing. Plasmid with either wild–type (wtLMW–PTP) or dominant negative (dnLMW–PTP) was transfected into B3 cells using lipofectamine. For determining the activation of ERK, cells (1.6 million) were gradually deprived serum, incubated in serum–free medium with/without 0.5 mM vanadate for 30 min before applying PDGF (1 ng/ml), and analyzed for phosphorylated ERK. Results: Both B3 and SRA01/04 cells showed strong total PTP activity by using either pNPP or phosphotyrosine as substrate. This activity was inhibited by vanadate but not by okadaic acid (an inhibitor for protein serine/threonine phosphatase). ERK was found to be transiently activated in normal B3 cells upon PDGF stimulation with maximum at 15 min. Cells pretreated with vanadate (0.5 mM, 30 min) abolished the PDGF–induced transient activation of ERK and increased the basal P–ERK in unstimulated cells. LMW–PTP in cell lysate was detected by immunoblot. Three different splicing forms of LMW–PTP cDNA were isolated using RT–PCR. Two of them (545 bp) were identical to human acid phosphatase ACP1 and ACP2. The third one (431 bp) was the new splicing form isolated for the first time. Increased amount of LMW–PTP was detected by western blot in B3 cells transfected with either wtLMW–PTP or dnLMW–PTP. Compared to control cells, transfection with wtLMW–PTP increased cellular total PTP activity about 30% while transfection with dnLMW–PTP decreased the activity about 20%. Conclusions: LMW–PTP is present in human lens epithelial cells and may regulate PDGF–induced signaling in lens epithelial cells.

Keywords: gene/expression • signal transduction