Abstract
There are more than 600 receptor-like kinases (RLKs) in Arabidopsis, but due to challenges associated with the characterization of membrane proteins, only a few have known biological functions. The plant RLK FERONIA is a peptide receptor and has been implicated in plant growth regulation, but little is known about its molecular mechanism of action. To investigate the properties of this enzyme, we used a cell-free wheat germ-based expression system in which mRNA encoding FERONIA was co-expressed with mRNA encoding the membrane scaffold protein variant MSP1D1. With the addition of the lipid cardiolipin, assembly of these proteins into nanodiscs was initiated. FERONIA protein kinase activity in nanodiscs was higher than that of soluble protein and comparable with other heterologously expressed protein kinases. Truncation experiments revealed that the cytoplasmic juxtamembrane domain is necessary for maximal FERONIA activity, whereas the transmembrane domain is inhibitory. An ATP analogue that reacts with lysine residues inhibited catalytic activity and labeled four lysines; mutagenesis demonstrated that two of these, Lys-565 and Lys-663, coordinate ATP in the active site. Mass spectrometric phosphoproteomic measurements further identified phosphorylation sites that were examined using phosphomimetic mutagenesis. The results of these experiments are consistent with a model in which kinase-mediated phosphorylation within the C-terminal region is inhibitory and regulates catalytic activity. These data represent a step further toward understanding the molecular basis for the protein kinase catalytic activity of FERONIA and show promise for future characterization of eukaryotic membrane proteins.
Highlights
The Arabidopsis receptor-like kinase (RLK)2 family currently contains more than 600 members and makes up 2.5% of the
To test whether the low specific activity reflected a possible requirement for FERONIA to be embedded within a lipid bilayer, we explored the use of nanodisc technology in combination with cell-free expression [19, 25]
We found that nonaffinity-tagged FERONIA co-expressed with StrepII-tagged MSP1D1 has significantly less background following StrepTactin purification than C-terminally His-tagged FERONIA subjected to immobilized nickel affinity purification (Fig. 1A)
Summary
The first attempt to produce FERONIA was made using a previously described dialysis cup cell-free translation technique [24]. Full-length FERONIA and the truncations TM and KD had significantly reduced activity compared with the JM truncation (Fig. 3C) These results indicate important roles for the TM and ecto domains in regulating the kinase catalytic activity, we cannot rule out other more trivial interpretations, such as TM-mediated aggregation that may be occurring in the absence of cardiolipin and causing steric inhibition of kinase activity. The Y495F mutation, located within the JM domain, resulted in a minor increase in kinase activity (Fig. 5) These observations suggest that C-terminal FERONIA phosphorylation may be involved in regulation of the kinase via inhibition in vivo, whereas Ser and Thr residues within the kinase domain may be important for structural stability of the protein. Tyr-495 phosphorylation may have some role in inhibition, indirect structural changes resulting from Tyr to Phe mutation cannot be ruled out
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