Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity.

Jens Hartmann,Margret Sauter,Dennis Linke,Andreas Tholey,Christine Bönniger

doi:10.1042/bj20150147

Abstract

PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser(696) and Ser(698) in the JM (juxtamembrane) region and probably Ser(886) and/or Ser(893) in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser(717) in the JM, and at Ser(733), Thr(752), Ser(783), Ser(864), Ser(911), Ser(958) and Thr(998) in the kinase domain. The LC-ESI-MS/MS spectra provided support that up to three sites (Thr(890), Ser(893) and Thr(894)) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr(890), Ser(893), Thr(894) and Thr(899), differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response.

Highlights

The pentapeptide PSK with the amino acid backbone YIYTQ has been described as a peptide hormone that promotes plant growth through elevated cell expansion
PSKR1 and PSKR2 belong to a large monophyletic gene family of Arabidopsis LRR leucine-rich repeat receptor-like kinase (RLK) [9,10,11]
LRR RLKs are characterized by a defined organization of functional domains: LRRs with an extracellular ligand-binding domain, a single transmembrane domain and a cytoplasmic protein portion consisting of a catalytic kinase domain (KD) flanked by two regulatory sequences, the JM region and a CT (C-terminal) domain

Summary

Introduction

The pentapeptide PSK (phytosulfokine) with the amino acid backbone YIYTQ has been described as a peptide hormone that promotes plant growth through elevated cell expansion. We used LC–ESI–MS/MS analysis of the recombinant PSKR1 KD to show that the cytoplasmic protein portion of the receptor is autophosphorylated on specific serine and threonine residues in vitro. We studied further the functional role of identified phosphorylation sites in the PSKR1 AL with respect to in vitro kinase activity.

Results

Conclusion