Characterization of Drosophila Protein Tyrosine Phosphatase PTP61F: Determination of Substrate Specificity by Subcellular Localization of PTP Isoforms
Date Issued
2007
Date
2007
Author(s)
Lee, Han
DOI
zh-TW
Abstract
The Drosophila protein tyrosine phosphatase dPTP61F is an ortholog of human PTP1B and TCPTP, both of which are involved in the regulation of various signaling pathways. Thus, it is essential to characterize the function of dPTP61F for revealing the potential role of PTPs in the control of signal transcution and development. The dPTP61F gene undergoes alternative splicing at 3’-end of the transcript, resulting in an internal membrane-associated form dPTP61Fm and a nucleus-localized form dPTP61Fn. These two PTP isoforms share the identical catalytic domain, suggesting that the substrate specificity may be modulated by the subcellular location of the phosphatase. In the current study, we have explored the functional roles of both two isoforms of dPTP61F.
Applying RNA interference (RNAi) approach to knock down both isoforms of dPTP61F, we found that the tyrosine phosphorylation of dIR was enhanced in S2 cells stimulated with insulin, suggesting that dPTP61F may function as a negative regulator in insulin-mediated signal transduction. Moreover, when overexpressed in S2 cells, only the dPTP61Fm, but not the dPTP61Fn, was capable of dephosphorylating dIR. Furthermore, we showed that the access of dIR was determined by the distinct cytosolic/plasma membrane localization of dPTP61Fm, as confirmed by results from immunofluorescence staining in S2 cells.
We further explored the potential role of the nuclear form of dPTP61F. It has been shown that the evolutionarily conserved ortholog of dPTP61Fn, the mammalian TCPTP, which dephosphorylates and inactivates transcription factor STAT, is present in nucleus. Therefore, we proposed that dPTP61Fn may antagonize JAK/STAT signaling by facilitating dephosphorylation of Drosophila STAT92E. In contrast, dPTP61Fm may exert a similar role as mammalian PTP1B, which recognizes JAK (Hop) as a potential substrate.
We observed that, when dPTP61Fm was overexpressed, Hop was significantly dephosphorylated. Forced expression of phosphatase-dead (C/S) mutant form of dPTP61Fm showed dominant negative effect. These results suggest that dPTP61Fm is indeed a Hop phosphatase and that the phosphorylation level of Hop is constitutively suppressed by the endogenous dPTP61Fm. Furthermore, the dephosphorylation event is executed only by the membrane-bound PTP isoform. In addition, we also found that the phosphorylation level of STAT92E was decreased when dPTP61Fn was overexpressed, suggesting that STAT92E is a potential substrate of the nuclear form of PTP. Interestingly, the protein expression of dPTP61F was increased in response to activation of Upd signaling following a time-dependent manner. Using the RNAi approach, we demonstrated that the up-regulation of dPTP61F gene expression was modulated by the transcription activity of STAT92E. Performing a RT-PCR experiment, we also confirmed that both the amount of dPTP61Fm and dPTP61Fn tarnscripts were affected by JAK/STAT activity. These results implicated that dPTP61F is a putative target gene of STAT and that both isoforms of this phosphatase may participate in the negative feedback regulation of JAK/STAT signaling pathway.
In summary, our studies have revealed the negative regulatory role of dPTP61F in Drosophila insulin and JAK/STAT signaling pathways. We showed that each of the dPTP61F isoform has specific functions in different compartments inside a single cell. Both pathways control important signaling events, suggesting that dPTP61F may play an essential role for the maintanance of proper cellular processes during Drosophila development.
Subjects
果蠅酪氨酸去磷酸酶
dPTP61F
受質專一性
Drosophila protein tyrosine phosphatase
substrate specificity
Type
other