title: p21-activated Kinases (PAKs) Mediate the Phosphorylation of PREX2 Protein to Initiate Feedback Inhibition of Rac1 GTPase.
abstract: Phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchanger 2 (PREX2) is a guanine nucleotide exchange factor (GEF) for the Ras-related C3 botulinum toxin substrate 1 (Rac1) GTPase, facilitating the exchange of GDP for GTP on Rac1. GTP-bound Rac1 then activates its downstream effectors, including p21-activated kinases (PAKs). PREX2 and Rac1 are frequently mutated in cancer and have key roles within the insulin-signaling pathway. Rac1 can be inactivated by multiple mechanisms; however, negative regulation by insulin is not well understood. Here, we show that in response to being activated after insulin stimulation, Rac1 initiates its own inactivation by decreasing PREX2 GEF activity. Following PREX2-mediated activation of Rac1 by the second messengers PIP3 or Gβγ, we found that PREX2 was phosphorylated through a PAK-dependent mechanism. PAK-mediated phosphorylation of PREX2 reduced GEF activity toward Rac1 by inhibiting PREX2 binding to PIP3 and Gβγ. Cell fractionation experiments also revealed that phosphorylation prevented PREX2 from localizing to the cellular membrane. Furthermore, the onset of insulin-induced phosphorylation of PREX2 was delayed compared with AKT. Altogether, we propose that second messengers activate the Rac1 signal, which sets in motion a cascade whereby PAKs phosphorylate and negatively regulate PREX2 to decrease Rac1 activation. This type of regulation would allow for transient activation of the PREX2-Rac1 signal and may be relevant in multiple physiological processes, including diseases such as diabetes and cancer when insulin signaling is chronically activated.
authors.affiliation: From the Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029, the Department of Pharmacology, Columbia University, New York, New York 10032.
authors.forename: Douglas
authors.initials: D
authors.lastname: Barrows
authors.affiliation: From the Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Schoenfeld
authors.affiliation: From the Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029.
authors.forename: Cindy
authors.initials: C
authors.lastname: Hodakoski
authors.affiliation: the Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University, New York, New York 10032.
authors.forename: Antonina
authors.initials: A
authors.lastname: Silkov
authors.affiliation: the Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University, New York, New York 10032.
authors.forename: Barry
authors.initials: B
authors.lastname: Honig
authors.affiliation: Cell Signaling Technology, Danvers, Massachusetts 01923.
authors.forename: Anthony
authors.initials: A
authors.lastname: Couvillon
authors.affiliation: the Department of Pharmacology and Experimental Therapy, Institute of Experimental and Clinical Pharmacology and Toxicology, Eberhard Karls University Hospitals and Clinics, and Interfaculty Center of Pharmacogenomics and Pharmaceutical Research, University of Tübingen, 72074 Tübingen, Germany.
authors.forename: Aliaksei
authors.initials: A
authors.lastname: Shymanets
authors.affiliation: the Department of Pharmacology and Experimental Therapy, Institute of Experimental and Clinical Pharmacology and Toxicology, Eberhard Karls University Hospitals and Clinics, and Interfaculty Center of Pharmacogenomics and Pharmaceutical Research, University of Tübingen, 72074 Tübingen, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Nürnberg
authors.affiliation: the Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02115, and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: John M
authors.initials: JM
authors.lastname: Asara
authors.affiliation: From the Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029, ramon.parsons@mssm.edu.
authors.forename: Ramon
authors.initials: R
authors.lastname: Parsons
mesh_terms: D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D010766:Phosphorylation / Q000502:physiology; D015290:Second Messenger Systems / Q000502:physiology*; D054462:p21-Activated Kinases / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D020662:Guanine Nucleotide Exchange Factors; C490712:PREX2 protein, human; C107953:RAC1 protein, human; D054462:p21-Activated Kinases; D020830:rac1 GTP-Binding Protein
keywords: G-protein-coupled receptor (GPCR); Phosphatidylinositol-3,4,5-trisphosphate-dependent RAC exchanger 2 (PREX2); Rac (Rac GTPase); guanine nucleotide exchange factor (GEF); insulin; phosphatidylinositide 3-kinase (PI 3-kinase); serine/threonine-protein kinase PAK 1 (PAK1); signal transduction