The Phytosulfokine (PSK) Receptor Is Capable of Guanylate Cyclase Activity and Enabling Cyclic GMP-dependent Signaling in Plants

Lusisizwe Kwezi,Oziniel Ruzvidzo,Janet I Wheeler,Kershini Govender,Sylvana Iacuone,Philip E Thompson,Chris Gehring,Helen R Irving

doi:10.1074/jbc.m110.168823

Abstract

Phytosulfokines (PSKs) are sulfated pentapeptides that stimulate plant growth and differentiation mediated by the PSK receptor (PSKR1), which is a leucine-rich repeat receptor-like kinase. We identified a putative guanylate cyclase (GC) catalytic center in PSKR1 that is embedded within the kinase domain and hypothesized that the GC works in conjunction with the kinase in downstream PSK signaling. We expressed the recombinant complete kinase (cytoplasmic) domain of AtPSKR1 and show that it has serine/threonine kinase activity using the Ser/Thr peptide 1 as a substrate with an approximate K(m) of 7.5 μm and V(max) of 1800 nmol min(-1) mg(-1) of protein. This same recombinant protein also has GC activity in vitro that is dependent on the presence of either Mg(2+) or Mn(2+). Overexpression of the full-length AtPSKR1 receptor in Arabidopsis leaf protoplasts raised the endogenous basal cGMP levels over 20-fold, indicating that the receptor has GC activity in vivo. In addition, PSK-α itself, but not the non-sulfated backbone, induces rapid increases in cGMP levels in protoplasts. Together these results indicate that the PSKR1 contains dual GC and kinase catalytic activities that operate in vivo and that this receptor constitutes a novel class of enzymes with overlapping catalytic domains.

Highlights

Guanylate cyclases (GCs)3 (EC 4.6.1.2) are the enzymes that generate production of cyclic GMP, which is a key signaling intermediate in eukaryotes
In Vitro GC Activity of AtPSKR1—The domain organization of PSKR1 is typical of leucine-rich repeat receptor-like kinase (LRR RLK) with a highly conserved leucine-rich repeat domain in the extracellular space, a transmembrane domain, and a kinase domain that forms the majority of the intracellular protein
We demonstrated that the BRI1 receptor contained a functional GC domain within its kinase domain [8], which raised the intriguing possibility that this GC domain contributed to downstream signaling

Summary

Introduction

Guanylate cyclases (GCs)3 (EC 4.6.1.2) are the enzymes that generate production of cyclic GMP (cGMP), which is a key signaling intermediate in eukaryotes. The recombinant protein was expressed as a His-tagged SUMO fusion protein in BL21 (DE3) E. coli cells and has a predicted molecular mass of 50.3 kDa. To test whether the GC search motif present was essential for the observed GC activity, we mutated the glycine residue in position 3 of the search motif to a lysine residue (Fig. 1D) as the glycine is predicted to face the purine and determine substrate specificity for GTP rather than ATP [33,34,35].

Results

Conclusion