Premium
Molecular characterization and phosphorylation site mapping of the Arabidopsis CRINKLY4 receptor‐like kinase (ACR4)
Author(s) -
Shah Shweta,
Meyer Matthew,
Rao A
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.724.18
Subject(s) - autophosphorylation , phosphorylation , protein kinase domain , biophysics , chemistry , kinase , biochemistry , extracellular , intracellular , biology , microbiology and biotechnology , protein kinase a , mutant , gene
ACR4 is required for overall growth and development of the plant. ACR4 architecture possesses an extracellular ligand binding domain, a transmembrane (TM) helix, and an intracellular domain (ICD). Previously, we have characterized the biochemical and biophysical properties of the ICD independent of the TM domain (Meyer et al., Biochemistry. 2011, 50:2170). However, the molecular association of the TM domain with plasma membrane and its effect on the functioning of ICD domain is still poorly understood. The main objective of this study is to characterize ACR4 ICD in association with the TM domain. We have recombinantly expressed soluble protein with a N‐terminal 6H‐SUMO tag and the TM domain followed by ICD (SUMO‐TM‐ICD). Gel filtration experiments suggest that the protein is a monomer. The far UV‐CD spectrum shows minima at 208 nm and 222 nm, characteristic of alpha helical structures, indicating that the protein is properly folded. We have also carried out a comprehensive mapping of the TM‐ICD autophosphorylation sites. A total of 20 phosphorylation sites were identified. Significantly, among the 20 phosphorylation sites, two occurred at tyrosine residues, suggesting that in vitro the kinase activity of ACR4 may not be restricted to Ser/Thr residues. Furthermore, in order to mimic the natural membrane bound environment, we have prepared SUMO‐TM‐ICD Nanodiscs. The SUMO‐TM‐ICD Nanodiscs were characterized by TEM and were found to be ~20 nm in size. SUMO‐TM‐ICD Nanodisc remains functionally active, affording great potential for further characterization of SUMO‐TM‐ICD in its near‐native environment.