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

Matthew R. Meyer, Cheryl F. Lichti, R. Reid Townsend, A. Gururaj Rao

Research output: Contribution to journalArticle

19 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)2170-2186
Number of pages17
JournalBiochemistry
Volume50
Issue number12
DOIs
StatePublished - Mar 29 2011
Externally publishedYes

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Oligomerization
Phosphorylation
Arabidopsis
Conformations
Phosphotransferases
Thermodynamic properties
Cytology
Proteolysis
Protein-Serine-Threonine Kinases
Divalent Cations
Fluorescence spectroscopy
Chromatography
Sedimentation
Recombinant Proteins
Trypsin
Escherichia coli
Mass spectrometry
Proteins
Plant Development
Fusion reactions

ASJC Scopus subject areas

  • Biochemistry

Cite this

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. / Meyer, Matthew R.; Lichti, Cheryl F.; Townsend, R. Reid; Rao, A. Gururaj.

In: Biochemistry, Vol. 50, No. 12, 29.03.2011, p. 2170-2186.

Research output: Contribution to journalArticle

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