Phosphoprotein and phosphopeptide interactions with the FHA domain from Arabidopsis kinase-associated protein phosphatase.
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.
Ding
Zhaofeng
Z
Department of Biochemistry, Division of Biological Sciences and Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65211, USA.
Wang
Huachun
H
Liang
Xiangyang
X
Morris
Erin R
ER
Gallazzi
Fabio
F
Pandit
Shashi
S
Skolnick
Jeffrey
J
Walker
John C
JC
Van Doren
Steven R
SR
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
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