A simple in vitro method to measure autophosphorylation of protein kinases
Tóm tắt
Receptor-like protein kinases (RLKs) are a large and important group of plant proteins involved in numerous aspects of development and stress response. Within this family, homo-oligermization of receptors followed by autophosphorylation of the intracellular protein kinase domain appears to be a widespread mechanism to regulate protein kinase activity. In vitro studies of several RLKs have identified autophosphorylation sites involved in regulation of catalytic activity and signaling in vivo. Recent work has established that multiple RLKs are biochemically active when expressed in E. coli and readily autophosphorylate prior to purification or subsequent manipulation. This observation has led us to develop a simplified method for assaying RLK phosphorylation status as an indirect measure of intrinsic autophosphorylation activity. The method involves expressing a recombinant RLK protein kinase domain in E. coli, followed by SDS-PAGE of boiled cell lysate, and sequential staining with the phosphoprotein stain Pro-Q Diamond and a colloidal Coomassie total protein stain. We show this method can be used to measure and quantify in vitro autophosphorylation levels of recombinant wildtype and mutant versions of the Arabidopsis RLK HAESA, as well as to detect transphosphorylation activity of recombinant HAESA against a protein kinase inactive version of itself. Our method has several advantages over traditional protein kinase assays. It does not require protein purification, transfer, blotting, or radioactive reagents. It allows for rapid and quantitative assessment of autophosphorylation levels and should have general utility in the study of any autophosphorylating protein kinase expressed in E. coli.
Tài liệu tham khảo
Horn MA, Walker JC: Biochemical properties of the autophosphorylation of RLK5, a receptor-like protein kinase from Arabidopsis thaliana. Biochim Biophys Acta. 1994, 1208: 65-74. 10.1016/0167-4838(94)90160-0.
Oh MH, Ray WK, Huber SC, Asara JM, Gage DA, Clouse SD: Recombinant brassinosteroid insensitive 1 receptor-like kinase autophosphorylates on serine and threonine residues and phosphorylates a conserved peptide motif in vitro. Plant Physiol. 2000, 124: 751-766. 10.1104/pp.124.2.751.
Wang X, Li X, Meisenhelder J, Hunter T, Yoshida S, Asami T, Chory J: Autoregulation and homodimerization are involved in the activation of the plant steroid receptor BRI1. Dev Cell. 2005, 8: 855-865. 10.1016/j.devcel.2005.05.001.
Hink MA, Shah K, Russinova E, de Vries SC, Visser AJWG: Fluorescence fluctuation analysis of Arabidopsis thaliana somatic embryogenesis receptor-like kinase and brassinosteroid insensitive 1 receptor oligomerization. Biophys J. 2008, 94: 1052-1062. 10.1529/biophysj.107.112003.
Wang X, Kota U, He K, Blackburn K, Li J, Goshe MB, Huber SC, Clouse SD: Sequential transphosphorylation of the BRI1/BAK1 receptor kinase complex impacts early events in brassinosteroid signaling. Dev Cell. 2008, 15: 220-235. 10.1016/j.devcel.2008.06.011.
Wang X, Goshe MB, Soderblom EJ, Phinney BS, Kuchar JA, Li J, Asami T, Yoshida S, Huber SC, Clouse SD: Identification and functional analysis of in vivo phosphorylation sites of the Arabidopsis BRASSINOSTEROID-INSENSITIVE1 receptor kinase. Plant Cell. 2005, 17: 1685-1703. 10.1105/tpc.105.031393.
Oh M-H, Wang X, Kota U, Goshe MB, Clouse SD, Huber SC: Tyrosine phosphorylation of the BRI1 receptor kinase emerges as a component of brassinosteroid signaling in Arabidopsis. Proc Natl Acad Sci USA. 2009, 106: 658-663. 10.1073/pnas.0810249106.
Oh M-H, Clouse SD, Huber SC: Tyrosine Phosphorylation of the BRI1 Receptor Kinase Occurs via a Post-Translational Modification and is Activated by the Juxtamembrane Domain. Front Plant Sci. 2012, 3: 175-
Oh M-H, Kim HS, Wu X, Clouse SD, Zielinski RE, Huber SC: Calcium/calmodulin inhibition of the Arabidopsis BRASSINOSTEROID-INSENSITIVE 1 receptor kinase provides a possible link between calcium and brassinosteroid signalling. Biochem J. 2012, 443: 515-523. 10.1042/BJ20111871.
Candiano G, Bruschi M, Musante L, Santucci L, Ghiggeri GM, Carnemolla B, Orecchia P, Zardi L, Righetti PG: Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis. 2004, 25: 1327-1333. 10.1002/elps.200305844.
Nolen B, Taylor S, Ghosh G: Regulation of protein kinases; controlling activity through activation segment conformation. Mol Cell. 2004, 15: 661-675. 10.1016/j.molcel.2004.08.024.
Agrawal GK, Thelen JJ: Development of a simplified, economical polyacrylamide gel staining protocol for phosphoproteins. Proteomics. 2005, 5: 4684-4688. 10.1002/pmic.200500021.