细胞生物学
生物化学
化学
磷酸化
激酶
突变体
自磷酸化
蛋白激酶A
生物
丝裂原活化蛋白激酶激酶
变构调节
MAP激酶激酶激酶
蛋白激酶结构域
作者
Matthew R. Meyer,Cheryl F. Lichti,R. Reid Townsend,A. Gururaj Rao
出处
期刊:Biochemistry
[American Chemical Society]
日期:2011-03-29
卷期号:50 (12): 2170-2186
被引量:25
摘要
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.
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