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.

Plant Receptors Go Endosomal: A Moving View on Signal Transduction

“…Furthermore, the protein phosphatase 2A inhibitor cantharidin affected FLS2 subcellular trafficking (Serrano et al, 2007). FLS2 was shown to interact with KAPP, a kinase interacting domain containing protein phosphatase 2C, as was also found for BAK1, BRI1, CLV1, SERK1, and SRK (Trotochaud et al, 1999;Gomez-Gomez et al, 2001;Shah et al, 2002;Vanoosthuyse et al, 2003;Ding et al, 2007). This indicates that KAPP keeps multiple RLKs under control by dephosphorylation.…”

The Arabidopsis Leucine-Rich Repeat Receptor Kinase BIR3 Negatively Regulates BAK1 Receptor Complex Formation and Stabilizes BAK1

“…401 BAK1 is a central co-receptor of several LRR-RKs and RLPs and is therefore an ideal 402 target for the regulation of multiple signaling pathways. Protein phosphatases (PP) 403 such as PP2A (Segonzac et al, 2014) and KINASE ASSOCIATED PROTEIN 404 PHOSPHATASE (KAPP) (Ding et al, 2007) negatively regulate BAK1 downstream 405 signaling, likely by dephosphorylating BAK1 or the interacting RKs, respectively. 406…”

Receptor Kinases in Plant Meristem Development

“…Another CLV signalling component is KAPP, a kinase-associated protein phosphatase that binds to phosphorylated peptides and serves to antagonize diverse RLKs in Arabidopsis Stone et al 1998;Ding et al 2007). Interaction studies and phosphorylation assays suggest that the MAP kinase pathway is involved in transmitting the CLV3 signal to the nucleus (Betsuyaku et al 2011).…”