title: Identification of in vitro autophosphorylation sites and effects of phosphorylation on the Arabidopsis CRINKLY4 (ACR4) receptor-like kinase intracellular domain: insights into conformation, oligomerization, and activity.
abstract: Arabidopsis CRINKLY4 (ACR4) is a receptor-like kinase (RLK) that consists of an extracellular domain and an intracellular domain (ICD) with serine/threonine kinase activity. While genetic and cell biology experiments have demonstrated that ACR4 is important in cell fate specification and overall development of the plant, little is known about the biochemical properties of the kinase domain and the mechanisms that underlie the overall function of the receptor. To complement in planta studies of the function of ACR4, we have expressed the ICD in Escherichia coli as a soluble C-terminal fusion to the N-utilization substance A (NusA) protein, purified the recombinant protein, and characterized the enzymatic and conformational properties. The protein autophosphorylates via an intramolecular mechanism, prefers Mn(2+) over Mg(2+) as the divalent cation, and displays typical Michaelis-Menten kinetics with respect to ATP with an apparent K(m) of 6.67 ± 2.07 μM and a V(max) of 1.83 ± 0.18 nmol min(-1) mg(-1). Autophosphorylation is accompanied by a conformational change as demonstrated by circular dichroism, fluorescence spectroscopy, and limited proteolysis with trypsin. Analysis by nanoliquid chromatography and mass spectrometry revealed 16 confirmed sites of phosphorylation at Ser and Thr residues. Sedimentation velocity and gel filtration experiments indicate that the ICD has a propensity to oligomerize and that this property is lost upon autophosphorylation.
authors.affiliation: Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa 50011, United States.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Meyer
authors.forename: Cheryl F
authors.initials: CF
authors.lastname: Lichti
authors.forename: R Reid
authors.initials: RR
authors.lastname: Townsend
authors.forename: A Gururaj
authors.initials: AG
authors.lastname: Rao
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000166:cytology / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D029968:Escherichia coli Proteins / Q000378:metabolism; D042541:Intracellular Space / Q000201:enzymology*; D008969:Molecular Sequence Data; D010445:Peptide Elongation Factors / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D055503:Protein Multimerization; D017346:Protein Serine-Threonine Kinases; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D035602:Transcriptional Elongation Factors
chemical_list: D029681:Arabidopsis Proteins; D029968:Escherichia coli Proteins; D010445:Peptide Elongation Factors; D011956:Receptors, Cell Surface; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C068727:nusA protein, E coli; D011494:Protein Kinases; C462019:ACR4 protein, Arabidopsis; D017346:Protein Serine-Threonine Kinases