RdoA is a eukaryotic-like serine/threonine protein kinase found in Salmonella typhimurium. It is a downstream effector of the Cpx stress response pathway and has been phenotypically characterized to have a functional role in flagellin phase variation and long-term bacterial survivability. Structurally, RdoA is homologous to, choline kinase and aminoglycoside (3’) phosphotransferase IIIa (APH[3’]IIIa). These kinases all belong to a protein kinase superfamily and share highly conserved residues/motifs in their catalytic domain. In RdoA seven of these conserved amino acids were proposed to have functional roles in the phosphotransfer mechanism. Mutation of these proposed catalytic domain residues resulted in a loss of in vitro kinase activity and in vivo RdoA function for a majority of the mutants. Four of the mutants also exhibited decreased levels of stable RdoA compared to wildtype.
Many protein kinases regulate activity through phosphorylation of an activation loop. Although RdoA does not contain a canonical activation loop, its carboxyl terminus is proposed to play a similar regulatory function. Mutations of a putative autophosphorylation target in the carboxyl terminus resulted in loss of in vitro kinase activity. Truncations of this region also resulted in loss of kinase activity, as well as decreasing RdoA stability. The length of the carboxyl terminus in the kinase was shown to be an important determinant in the overall structural stability of RdoA. Mutational analyses of conserved amino acid residues surrounding the putative substrate-binding cleft of RdoA revealed site specific mutants with diminished in vitro phosphorylation activity and/or RdoA levels. A subset of these mutants for which no in vitro kinase activity was detected were still able to complement RdoA function in vivo. Taken together these results indicate that this region of the protein is important for RdoA function.
In summary, this work has generated a panel of RdoA mutants with several unique phenotypes that will facilitate characterization of RdoA function and of regions of the protein / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2010-09-29 21:35:42.815
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OKQ.1974/6118 |
| Date | 30 September 2010 |
| Creators | LIN, JANET TING-MEI |
| Contributors | Queen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.)) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner. |
| Relation | Canadian theses |
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