Kinase activity and calmodulin binding are essential for growth signaling by the phytosulfokine receptor PSKR1

Abstract

SUMMARYThe cell growth-promoting peptide phytosulfokine (PSK) is perceived by leucine-rich repeat (LRR) receptor kinases. To elucidate PSK receptor function we analyzed PSKR1 kinase activity and binding to Ca 2+ sensors and evaluated the contribution of these activities to growth control in planta. Ectopically expressed PSKR1 was capable of auto-and transphosphorylation. Replacement of a conserved lysine within the ATP-binding region by a glutamate resulted in the inhibition of auto-and transphosphorylation kinase activities. Expression of the kinase-inactive PSKR1(K762E) receptor in the pskr null background did not restore root or shoot growth. Instead, the mutant phenotype was enhanced suggesting that the inactive receptor protein exerts growth-inhibitory activity. Bioinformatic analysis predicted a putative calmodulin (CaM)-binding site within PSKR1 kinase subdomain VIa. Bimolecular fluorescence complementation analysis demonstrated that PSKR1 binds to all isoforms of CaM, more weakly to the CaM-like protein CML8 but apparently not to CML9. Mutation of a conserved tryptophan (W831S) within the predicted CaM-binding site strongly reduced CaM binding. Expression of PSKR1(W831S) in the pskr null background resulted in growth inhibition that was similar to that of the kinase-inactive receptor. We conclude that PSK signaling requires Ca 2+ /CaM binding and kinase activity of PSKR1 in planta. We further propose that the inactivated kinase interferes with other growth-promoting signaling pathway(s).

A Plant Phytosulfokine Peptide Initiates Auxin-Dependent Immunity through Cytosolic Ca2+ Signaling in Tomato

“…Calmodulins (CaMs) are Ca 2+ binding proteins whose affinities to target proteins, and hence activities, are modified upon Ca 2+ binding. This process translates changes in local [Ca 2+ ] into specific physiological responses (McCormack et al, 2005;Hartmann et al, 2014). In tomato, there are six homologous CaM genes encoding a total of four CaM protein isoforms.…”

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

“…The pskr1-3 T-DNA insertion line, the double knockout line pskr1-3 pskr2-1, and the 35S:PSKR1-GFP-1 line were previously described (Kutschmar et al, 2009;Stührwohldt et al, 2011;Hartmann et al, 2014). The cgnc17 T-DNA insertion line SALK_041923 and homozygous seeds for the BAK1 T-DNA insertion lines, bak1-3 (SALK_116202C), and bak1-4 (SALK_034523C) were obtained from NASC (Nottingham Arabidopsis Stock Centre, University of Nottingham, Nottingham, UK).…”

“…CNGC14, CNGC15, CNGC16, CNGC17, CBGC18, and ACTIN2 cDNAs as a loading control were amplified with the gene-specific primers listed in Supplemental Table 1. qPCR analysis of CNGC14, CNGC15, CNGC17, and CBGC18 was performed with 10 ng cDNA each using the Rotor-Gene SYBR Green PCR Kit (Qiagen) with ACTIN2 and GAPC1 as control genes using the primers listed in Supplemental Table 1. The relative transcript abundance was (Hartmann et al, 2014), and was described as a GC (Kwezi et al, 2011). PSKR1 interacts with BAK1 and with the H + -ATPases AHA1 and AHA2.…”

“…Downstream of perception, the PSK signaling pathway is not understood. Recent data describe binding of PSKR1 to calmodulin (CaM) (Hartmann et al, 2014). When a conserved tryptophan at the CaM binding site of PSKR1 was mutated, the receptor variant was no longer able to bind CaM.…”

“…A single transmembrane domain links the LRR region to a cytoplasmic kinase domain. The intracellular PSKR1 protein part was shown to possess serine/threonine kinase activity (Kwezi et al, 2011;Hartmann et al, 2014). The kinase domain of PSKR1 overlaps with a canonical guanylate cyclase (GC) core and was shown to also have, albeit weak, GC activity in vitro.…”