Analysis of Phosphorylation of the Receptor-Like Protein Kinase HAESA during Arabidopsis Floral Abscission

Abstract

Receptor-like protein kinases (RLKs) are the largest family of plant transmembrane signaling proteins. Here we present functional analysis of HAESA, an RLK that regulates floral organ abscission in Arabidopsis. Through in vitro and in vivo analysis of HAE phosphorylation, we provide evidence that a conserved phosphorylation site on a region of the HAE protein kinase domain known as the activation segment positively regulates HAE activity. Additional analysis has identified another putative activation segment phosphorylation site common to multiple RLKs that potentially modulates HAE activity. Comparative analysis suggests that phosphorylation of this second activation segment residue is an RLK specific adaptation that may regulate protein kinase activity and substrate specificity. A growing number of RLKs have been shown to exhibit biologically relevant dual specificity toward serine/threonine and tyrosine residues, but the mechanisms underlying dual specificity of RLKs are not well understood. We show that a phospho-mimetic mutant of both HAE activation segment residues exhibits enhanced tyrosine auto-phosphorylation in vitro, indicating phosphorylation of this residue may contribute to dual specificity of HAE. These results add to an emerging framework for understanding the mechanisms and evolution of regulation of RLK activity and substrate specificity.

Mechanistic insight into a peptide hormone signaling complex mediating floral organ abscission

“…Upon IDA binding at the cell surface, the kinase domains of HAESA and SERK1, which have been shown to be active protein kinases ( Jinn et al, 2000 ; Shah et al, 2001 ; Taylor et al, 2016 ), may interact in the cytoplasm to activate each other. Consistently, the HAESA kinase domain can transphosphorylate SERK1 and vice versa in in vitro transphosphorylation assays ( Figure 3E ).…”

Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination

“…In addition, the mutation S1044/5A in the BRI1 activation loop results in loss of function in BRI1 (Wang et al, 2005), as does the mutation T455A in the BAK1 (SERK3) activation loop . Furthermore, HAE S856A (in the activation loop), which has an abolished autophosphorylation activity, is capable of partially rescuing the hae hsl2 abscissiondeficient mutant (Taylor et al, 2016). Although the phosphorylation mimic HAE S856D-S861D maintains some autophosphorylation activity, it fails to complement the hae hsl2 phenotype.…”

Disrupting ER-associated protein degradation suppresses the abscission defect of a weak hae hsl2 mutant in Arabidopsis

“…T 1 s were selected on half-stength Murashige and Skoog (1/2 MS) plates (2.16g of MS basal medium per 1 liter, supplemented with 1% sucrose, w/v) containing 0.8% agar and kanamycin (K50), and then transplanted onto soil following selection. The HAEpr:HAE-YFP construct used was previously described ( Taylor et al , 2016 ). This construct was then mutagenized to introduce the C222Y mutation of hae-3 by the Quick-Change site-directed mutagenesis method (Agilent Technologies, Santa Clara, CA, USA).…”