Identification of Ligand Binding Site of Phytosulfokine Receptor by On-column Photoaffinity Labeling

Abstract

Phytosulfokine (PSK), an endogenous 5-amino-acid-secreted peptide in plants, affects cellular potential for growth via binding to PSKR1, a member of the leucine-rich repeat receptor kinase (LRR-RK) family. PSK interacts with PSKR1 in a highly specific manner with a nanomolar dissociation constant. However, it is not known which residues in the PSKR1 extracellular domain constitute the ligand binding pocket. Cell-to-cell communication is essential for growth and development of multicellular organisms throughout their life. In plants, hormones, including small lipophilic compounds and secreted peptides, comprise a large group of signaling molecules that are central to intercellular communication. They elicit biological activity by binding to cell surface receptors that have kinase activity or by directly interacting with intracellular proteins.Phytosulfokine (PSK) 2 is a 5-amino-acid-secreted peptide that has been identified in the medium of plant cell cultures, based on the results of assays of the growth-promoting activity of cultured cells (1). The addition of chemically synthesized PSK to culture medium, even at nanomolar concentrations, significantly promotes the proliferation of callus and suspension cells. PSK also promotes tracheary element differentiation (2), somatic embryogenesis (3, 4), adventitious bud formation (5), adventitious root formation (6), and pollen germination in vitro (7). PSK is produced from Ϸ80-amino-acid precursor peptides via post-translational sulfation of tyrosine residues and proteolytic processing (8). Genes encoding PSK precursors are redundantly distributed in the genome and are expressed in cultured cells and in a variety of tissues, including leaves, stems, flowers, and roots (9, 10). PSK binds the membrane-localized PSK receptor PSKR1, which is a leucine-rich repeat receptor kinase (LRR-RK) that has been purified from solubilized carrot microsomes by ligand-based affinity chromatography (hereafter referred to as DcPSKR1) (11). The extracellular domain of DcPSKR1 contains 21 tandem copies of LRR interrupted by a 36-amino-acid island domain rich in hydrophilic and charged amino acid residues. Disruption or overexpression of the Arabidopsis ortholog of PSKR1 (AtPSKR1) significantly alters cellular longevity and potential for growth without interfering with primary morphogenesis of plants (10). PSK appears to activate the basic potential for cellular growth rather than directly determining cell fate and thereby exerts a pleiotropic effect on individual cells in response to environmental hormonal conditions.Ligand binding generally causes a receptor protein to undergo a conformational change that directly activates the receptor so that it can interact with another cellular molecule and/or exert intrinsic enzyme activities such as kinase activity. PSK interacts with DcPSKR1 in a highly specific manner, with a high affinity dissociation constant of K d ϭ 4.2 nM (11). However, it is not known which amino acids in the DcPSKR1 extracellular domain constitute the ligand binding pocket....

Tyrosine-sulfated glycopeptide involved in cellular proliferation and expansion in Arabidopsis

“…Transformation of tobacco BY-2 cells was performed as described previously (24). Preparation of microsomal fractions of tobacco BY-2 cells and the photoaffinity labeling by using [ 125 I]ASA-PSK were performed as described previously (24). Affinity-purified antibodies were prepared as described previously (12).…”

“…For the overexpression of At1g72300 and At5g53890 in tobacco BY-2 cells, PCR fragments coding for At1g72300 and At5g53890 were ligated into the binary vector pBI121 by replacing the GUS-coding sequence downstream of the CaMV 35S promoter, respectively. Transformation of tobacco BY-2 cells was performed as described previously (24). Preparation of microsomal fractions of tobacco BY-2 cells and the photoaffinity labeling by using [ 125 I]ASA-PSK were performed as described previously (24).…”

Direct interaction of ligand–receptor pairs specifying stomatal patterning

“…1A-D). Thus, as with other LRRRKs, such as CLAVATA1 (CLV1) and PSKR (Shinohara et al 2007;Ogawa et al 2008), the kinase domain of ERECTA family proteins is not required for ligand binding. In contrast, TMM-GFP failed to coimmunoprecipitate EPF1-Flag (Fig.…”

Phytosulfokine Regulates Growth in Arabidopsis through a Response Module at the Plasma Membrane That Includes CYCLIC NUCLEOTIDE-GATED CHANNEL17, H+-ATPase, and BAK1

“…The leucine-rich repeats of PSKR1, PSKR2, and BRI1 contain an island domain that was shown to bind the respective ligands, i.e., PSK and brassinolide (Kinoshita et al, 2005;Shinohara et al, 2007;Clouse, 2011). A single transmembrane domain links the LRR region to a cytoplasmic kinase domain.…”