title: Glia maturation factor (GMF) interacts with Arp2/3 complex in a nucleotide state-dependent manner.
abstract: Glia maturation factor (GMF) is a member of the actin-depolymerizing factor (ADF)/cofilin family. ADF/cofilin promotes disassembly of aged actin filaments, whereas GMF interacts specifically with Arp2/3 complex at branch junctions and promotes debranching. A distinguishing feature of ADF/cofilin is that it binds tighter to ADP-bound than to ATP-bound monomeric or filamentous actin. The interaction is also regulated by phosphorylation at Ser-3 of mammalian cofilin, which inhibits binding to actin. However, it is unknown whether these two factors play a role in the interaction of GMF with Arp2/3 complex. Here we show using isothermal titration calorimetry that mammalian GMF has very low affinity for ATP-bound Arp2/3 complex but binds ADP-bound Arp2/3 complex with 0.7 μM affinity. The phosphomimetic mutation S2E in GMF inhibits this interaction. GMF does not bind monomeric ATP- or ADP-actin, confirming its specificity for Arp2/3 complex. We further show that mammalian Arp2/3 complex nucleation activated by the WCA region of the nucleation-promoting factor N-WASP is not affected by GMF, whereas nucleation activated by the WCA region of WAVE2 is slightly inhibited at high GMF concentrations. Together, the results suggest that GMF functions by a mechanism similar to that of other ADF/cofilin family members, displaying a preference for ADP-Arp2/3 complex and undergoing inhibition by phosphorylation of a serine residue near the N terminus. Arp2/3 complex nucleation occurs in the ATP state, and nucleotide hydrolysis promotes debranching, suggesting that the higher affinity of GMF for ADP-Arp2/3 complex plays a physiological role by promoting debranching of aged branch junctions without interfering with Arp2/3 complex nucleation.
authors.affiliation: From the Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Boczkowska
authors.affiliation: From the Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Rebowski
authors.affiliation: From the Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104. Electronic address: droberto@mail.med.upenn.edu.
authors.forename: Roberto
authors.initials: R
authors.lastname: Dominguez
mesh_terms: D051339:Actin Depolymerizing Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051376:Actin-Related Protein 2-3 Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000199:Actins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002417:Cattle; D020904:Glia Maturation Factor / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation / Q000235:genetics; D011485:Protein Binding / Q000502:physiology; D051300:Wiskott-Aldrich Syndrome Protein Family / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D051339:Actin Depolymerizing Factors; D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; D020904:Glia Maturation Factor; C120152:WASF2 protein, human; D051300:Wiskott-Aldrich Syndrome Protein Family; C117487:glia maturation factor gamma; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate
keywords: ADP; ATP; Actin; Arp2/3 Complex; GMF; Isothermal Titration Calorimetry; Protein Phosphorylation
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