Effects of Src Kinase Inhibition on Expression of Protein Tyrosine Phosphatase 1B after Brain Hypoxia in a Piglet Animal Model

Abstract

Background: Protein tyrosine phosphatases (PTPs) in conjunction with protein tyrosine kinases (PTKs) regulate cellular processes by posttranslational modifications of signal transduction proteins. PTP nonreceptor type 1B (PTP-1B) is an enzyme of the PTP family. We have previously shown that hypoxia induces an increase in activation of a class of nonreceptor PTK, the Src kinases. In the present study, we investigated the changes that occur in the expression of PTP-1B in the cytosolic component of the brain of newborn piglets acutely after hypoxia as well as long term for up to 2 weeks.

Methods: Newborn piglets were divided into groups: normoxia, hypoxia, hypoxia followed by 1 day and 15 days in FiO₂ 0.21, and hypoxia pretreated with Src kinase inhibitor PP2, prior to hypoxia followed by 1 day and 15 days. Hypoxia was achieved by providing 7% FiO₂ for 1 hour and PTP-1B expression was measured via immunoblotting.

Results: PTP-1B increased posthypoxia by about 30% and persisted for 2 weeks while Src kinase inhibition attenuated the expected PTP-1B-increased expression.

Conclusions: Our study suggests that Src kinase mediates a hypoxia-induced increased PTP-1B expression.

Figure 1

Experimental protocol.

Figure 2

(a) Immunoblotting was performed with an antibody specific for the PTP-1B. One band was identified at 50 kDa corresponding to the active PTP-1B. Results are expressed as OD values (mean ± SD). In this figure, also, we include data from piglets that were left to recover after 1 hour of hypoxia (for 1 day and for 15 days). (b) There was an increased expression of PTP-1B acutely after hypoxia that was maintained up to 15 days. Pretreatment with a Src inhibitor (PP2) did not affect the expression of PTP-1B in the acute phase 1 hour after hypoxia but blocked the expression of PTP-1B at 1 day posthypoxia. There was a trend for decreased PTP-1B expression at 15 days in piglets that were pretreated with PP2, but this did not reach statistical significance. Acute groups: ^∗P < 0.05 Hx and Hx+PP2 versus Nx. ^#NS Hx versus Hx+PP2. Recovery 1 day: ^&P < 0.05 Hx D1 versus Hx D1+PP2 and Nx. Recovery 15 days: ^‡NS Hx D15 versus Hx D15+PP2 and Nx.

Figure 3

(1–3) Hypoxia induces PTP-1B and Src kinase. In this paradigm, ROS are shown as a mediator for this effect. Inflammatory pathways could also culminate to the activation of Src and PTP-1B. (4, 5) Src kinase phosphorylates and interferes with PTP-1B action. Use of PP2 (an Src kinase inhibitor) would ameliorate the effects of Src kinase and hence would inhibit PTP-1B production as shown in this study. Nitric oxide synthase might also interfere in this pathway. NO production was increased in cells overexpressing PTP-1B and treatment with PP2 significantly decreased its production. (6) Src kinase has a negative regulatory phosphorylation site (tyrosine-527). PTP-1B is found to dephosphorylate Src kinase at this residue. Tyr-527 occupies the carboxy-proximal area of Src and phosphorylation at this site has been shown to deactivate the enzyme. PTP-1B dephosphorylates and activates Src. (7) Under different circumstances, PTPs may dephosphorylate specific components of the Src kinase-associated complexes and lead to an actual disassembly of those and loss of their activity.