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Biochemistry. 2007 Mar 13;46(10):2684-96. Epub 2007 Feb 16.

Phosphoprotein and phosphopeptide interactions with the FHA domain from Arabidopsis kinase-associated protein phosphatase.

Author information

1
Department of Biochemistry, Division of Biological Sciences and Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65211, USA.

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.

PMID:
17302430
DOI:
10.1021/bi061763n
[Indexed for MEDLINE]

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