title: Proteome analysis of erythrocytes lacking AMP-activated protein kinase reveals a role of PAK2 kinase in eryptosis.
abstract: Activation of AMP-activated protein kinase (AMPK) upon energy depletion stimulates energy production and limits energy utilization. Erythrocytes lacking AMPK are susceptible to suicidal cell death (eryptosis). A hallmark of eryptosis is cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface, which can be identified from annexin V-binding. AMPKα1-deficient mice (ampk(-/-)) suffer from anemia due to accelerated clearance of erythrocytes from circulating blood. To determine the link between AMPK and the eryptotic phenotype, we performed a global proteome analysis of erythrocytes from ampk(-/-) mice and wild-type mice using high-accuracy mass spectrometry and label-free quantitation and measured changes of expression levels of 812 proteins. Notably, the p21-activated kinase 2 (PAK2), previously implicated in apoptosis, was detected as downregulated in erythrocytes of ampk(-/-) mice, pointing to its potential role in eryptosis. To validate this, we showed that specific inactivation of PAK2 with the inhibitor IPA3 in human and murine ampk(+/+) erythrocytes increases the binding of annexin V and augments the stimulating effect of glucose deprivation on annexin V-binding. Inhibition of PAK2 failed to significantly modify annexin V-binding in ampk(-/-) erythrocytes, showing that AMPK and PAK2 exert similar phenotypes upon inactivation in erythrocytes. This study presents the first large-scale analysis of protein expression in erythrocytes from AMPKα1-deficient mice and reveals a role of PAK2 kinase in eryptosis.
authors.affiliation: Department of Physiology, University of Tübingen, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Zelenak
authors.forename: Michael
authors.initials: M
authors.lastname: Föller
authors.forename: Ana
authors.initials: A
authors.lastname: Velic
authors.forename: Karsten
authors.initials: K
authors.lastname: Krug
authors.forename: Syed M
authors.initials: SM
authors.lastname: Qadri
authors.forename: Benoit
authors.initials: B
authors.lastname: Viollet
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
authors.forename: Boris
authors.initials: B
authors.lastname: Macek
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016923:Cell Death / Q000502:physiology*; D004912:Erythrocytes / Q000737:chemistry* / Q000201:enzymology* / Q000502:physiology*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D018345:Mice, Knockout; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D054462:p21-Activated Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D020543:Proteome; D054462:p21-Activated Kinases; D055372:AMP-Activated Protein Kinases