Protein tyrosine phosphatase-PEST mediates hypoxia-induced endothelial autophagy and angiogenesis via AMPK activation

Abstract

Global and endothelial loss of PTP-PEST (also known as PTPN12) is associated with impaired cardiovascular development and embryonic lethality. Although hypoxia is implicated in vascular remodelling and angiogenesis, its effect on PTP-PEST remains unexplored. Here we report that hypoxia (1% oxygen) increases protein levels and catalytic activity of PTP-PEST in primary endothelial cells. Immunoprecipitation followed by mass spectrometry revealed that α subunits of AMPK (α1 and α2, encoded by PRKAA1 and PRKAA2, respectively) interact with PTP-PEST under normoxia but not in hypoxia. Co-immunoprecipitation experiments confirmed this observation and determined that AMPK α subunits interact with the catalytic domain of PTP-PEST. Knockdown of PTP-PEST abrogated hypoxia-mediated tyrosine dephosphorylation and activation of AMPK (Thr172 phosphorylation). Absence of PTP-PEST also blocked hypoxia-induced autophagy (LC3 degradation and puncta formation), which was rescued by the AMPK activator metformin (500 µM). Because endothelial autophagy is a prerequisite for angiogenesis, knockdown of PTP-PEST also attenuated endothelial cell migration and capillary tube formation, with autophagy inducer rapamycin (200 nM) rescuing angiogenesis. In conclusion, this work identifies for the first time that PTP-PEST is a regulator of hypoxia-induced AMPK activation and endothelial autophagy to promote angiogenesis.

In-silico identification of Tyr232 in AMPKα2 as a dephosphorylation site for the protein tyrosine phosphatase PTP-PEST

“…Whether such an increased binding results in altered enzymatic kinetics or can potentially lead to substrate trapping is an interesting future direction to investigate. Our earlier experimental studies have shown that AMPKα interacts with PTP-PEST under normoxic condition and that this interaction is impaired in hypoxia-treated cells 9 . The observations from the current simulations indicate an increased binding between AMPKα and PTP-PEST upon phosphorylation of Y64 on PTP-PEST.…”

“…It is a heterotrimeric enzyme consisting of α, β and γ subunits, with each subunit having different isoforms (two α, two β and three γ isoforms) 11 . The AMPKα subunit that interacts with PTP-PEST contains the kinase domain (KD) as well as the autoinhibitory domain (AID) 9 .…”

“…Using cellular and enzymatic approaches, we recently demonstrated for the first time that the catalytic domain of PTP-PEST interacts with the α subunits of AMPK (5'-adenosine monophosphate activated protein kinase) to mediate the tyrosine dephosphorylation and consequent activation of the latter 9 . AMPK is a serine/threonine kinase that plays an important role in cell metabolism, cellular stress response and autophagy 10 .…”

Autophagy and skin wound healing

“…Chandel et al. [ 58 ] confirmed that the protein tyrosine phosphatase, which contains proline-glutamate-serine-threonine as a potential major factor, is associated with inducing autophagy and angiogenesis by regulating hypoxia-activated AMPK signaling in endothelial cells.…”

Targeting Breast Cancer and Their Stem Cell Population through AMPK Activation: Novel Insights

“…There is increasing evidence suggesting the link between PTP inhibition and AMPK activation. Several biochemical consequences of PTP inhibition are believed to be executed via AMPK activation [149]. PTP1B is known to negatively modulate AMPK in peripheral tissues [33,150,151], and PTP1B deficiency is known to cause AMPK activation and autophagy [152].…”