title: Phosphoprotein and phosphopeptide interactions with the FHA domain from Arabidopsis kinase-associated protein phosphatase.
abstract: FHA domains are phosphoThr recognition modules found in diverse signaling proteins, including kinase-associated protein phosphatase (KAPP) from Arabidopsis thaliana. The kinase-interacting FHA domain (KI-FHA) of KAPP targets it to function as a negative regulator of some receptor-like kinase (RLK) signaling pathways important in plant development and environmental responses. To aid in the identification of potential binding sites for the KI-FHA domain, we predicted (i) the structure of a representative KAPP-binding RLK, CLAVATA1, and (ii) the functional surfaces of RLK kinase domains using evolutionary trace analysis. We selected phosphopeptides from KAPP-binding Arabidopsis RLKs for in vitro studies of association with KI-FHA from KAPP. Three phosphoThr peptide fragments from the kinase domain of CLV1 or BAK1 were found to bind KI-FHA with KD values of 8-20 microM, by NMR or titration calorimetry. Their affinity is driven by favorable enthalpy and solvation entropy gain. Mutagenesis of these three threonine sites suggests Thr546 in the C-lobe of the BAK1 kinase domain to be a principal but not sole site of KI-FHA binding in vitro. The brassinosteroid receptor BRI1 and KAPP are shown to associate in vivo and in vitro. Further genetic studies indicate that KAPP may be a negative regulator of the BRI1 signaling transduction pathway. 15N-Labeled KI-FHA was titrated with the GST-BRI1 kinase domain and monitored by NMR. BRI1 interacts with the same 3/4, 4/5, 6/7, 8/9, and 10/11 recognition loops of KI-FHA, with similar affinity as the phosphoThr peptides.
authors.affiliation: Department of Biochemistry, Division of Biological Sciences and Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65211, USA.
authors.forename: Zhaofeng
authors.initials: Z
authors.lastname: Ding
authors.forename: Huachun
authors.initials: H
authors.lastname: Wang
authors.forename: Xiangyang
authors.initials: X
authors.lastname: Liang
authors.forename: Erin R
authors.initials: ER
authors.lastname: Morris
authors.forename: Fabio
authors.initials: F
authors.lastname: Gallazzi
authors.forename: Shashi
authors.initials: S
authors.lastname: Pandit
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Skolnick
authors.forename: John C
authors.initials: JC
authors.lastname: Walker
authors.forename: Steven R
authors.initials: SR
authors.lastname: Van Doren
mesh_terms: D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D008565:Membrane Proteins; D008958:Models, Molecular; D010748:Phosphopeptides / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013912:Threonine / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D008565:Membrane Proteins; D010748:Phosphopeptides; D010750:Phosphoproteins; C083557:p112 protein, rat; D013912:Threonine; D011494:Protein Kinases; C108270:BRI1 protein, Arabidopsis; C090240:KAPP protein, Arabidopsis; D010749:Phosphoprotein Phosphatases