Plants possess integrated signalling networks that mediate the responses to various environmental conditions. Mitogen-activated protein kinases (MAPKs) constitute a highly conserved family of enzymes in eukaryotes, and in plants MAPK-based signal transduction modules regulate a large number of physiological processes, including responses to environmental stresses and phytohormones. Regulated dephosphorylation of active MAPKs is a key component of the control of MAPK signalling cascades, and in mammals, members of the MAPK phosphatase (MKP) sub-class of dual-specificity tyrosine phosphatases have been recognized as key players for inactivating MAPKs. Five MKP homologues are found in Arabidopsis thaliana, but only limited information is available concerning their properties and biological roles. Based on initial data derived from my reverse genetics and protein interaction studies of these five potential MKPs, as well as gene function information in the literature, I chose to focus on two putative Arabidopsis MKPs, AtMKP2 and Indole-3-Butyric Acid-response 5 (IBR5). By using a combination of genetic and biochemical studies, I established that the previously uncharacterized MKP designated AtMKP2, participates in the regulation of cellular homeostasis in ozone-challenged tissue, and can influence the activation state of two MAPKs, MPK3 and MPK6. AtMKP2-suppressed plants displayed significantly prolonged MPK3 and MPK6 activation during ozone treatment, and recombinant AtMKP2 was able to dephosphorylate both phospho-MPK3 and phospho-MPK6 in vitro, providing direct evidence that AtMKP2 may target these oxidant-activated MAPKs. A mutation in IBR5, one of the five potential AtMKPs, was previously reported to confer reduced sensitivity to auxin and ABA in Arabidopsis. My protein interaction studies demonstrated that IBR5 and MPK12 are physically coupled and that the C-terminus of MPK12 is essential for its interaction with IBR5. In vitro dephosphorylation assays indicated that recombinant phosphoMPK12 is efficiently dephosphorylated by IBR5. In transgenic plants with reduced expression of the MPK12 gene, root growth is hypersensitive to exogenous auxins, consistent with the lower auxin sensitivity reported for ibr5 mutants. Taken together, my data demonstrate for the first time that both AtMKP2 and IBR5 are bona fide Arabidopsis MAPK phosphatases and that they serve as important regulators of oxidative stress and auxin signalling, respectively, in Arabidopsis.

No description available.

Programmed cell death (PCD) is a fundamentally important process delicately coordinated throughout an organism’s life cycle. In plants, PCD is an integral part of development, reproduction, and pathogenesis. Numerous types of proteins are involved in regulation of PCD in plants, like phosphatases, metacaspases, and protein kinases. In tomato resistance to the pathogen Pseudomonas syringae pv. tomato (Pst), a Ser/Thr protein kinase, Adi3 (AvrPto-dependent Pto-interacting protein 3), interacts with the pathogen’s avirulence protein AvrPto and the tomato resistance protein Pto. Adi3 is a member of the AGC protein kinase family, a group known to transmit signals via the secondary messengers cAMP, cGMP, and phospholipids. In an unchallenged system, the master regulator of AGC kinases, Pdk1, activates Adi3. Activation of Adi3 enables nuclear localization and cell death suppression – all of which is prevented when challenged by Pst. A BLAST_p search of the Arabidopsis thaliania genome with the amino acid sequence of Adi3 identified a 67% identical match, AGC1-3. Like Adi3, AGC1-3 at its activation-loop serine and another site, by Pdk1. With N-terminal deletions of AGC1-3, Pdk1 was found to phosphorylate AGC1-3 at two serines – one serine conserved among all Arabidopsis AGC kinases, the other a serine on the N-terminus of the kinase domain. The non-activation loop serine in AGC1-3, Ser269, is conserved at Ser212, in Adi3. Phosphorylation at Ser212 does not impact auto-catalytic activity of Adi3. However it does enhance trans-catalytic activity. Analysis of AGC1-4 and AGC1-7, two proteins closely related to AGC1-3, reveals that phosphorylation of non-activation loop residues by Pdk1 is not restricted to AGC1-3 and Adi3. Functional analysis of AGC1-3 in Arabidopsis protoplasts revealed that like Adi3, nuclear localization and activation-loop phosphorylation are essential for cell viability. In an effort to elucidate a signaling network controlled by AGC1-3, the KiC (Kinase-client) assay was employed. In the KiC assay, a 2,100-member peptide library was assayed against AGC1-3 and the constitutively active mutant AGC1-3^(S596D). By MS analysis, AGC1-3 and AGC1-3(S596D) phosphorylated 26 and 19 substrate peptides, respectively. Substrate peptides were mapped to proteins involved in central metabolism, transcription, and protein metabolism. The work presented in this dissertation provides conclusive evidence that Pdk1 phosphorylates AGC1-3 and Adi3 at a non-activation loop residue. The work also supports AGC1-3 as the Arabidopsis homologue of Adi3 and presents novel phosphorylation data of potential AGC1-3 substrates.

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

No description available.

No description available.

Plants possess integrated signalling networks that mediate the responses to various environmental conditions. Mitogen-activated protein kinases (MAPKs) constitute a highly conserved family of enzymes in eukaryotes, and in plants MAPK-based signal transduction modules regulate a large number of physiological processes, including responses to environmental stresses and phytohormones. Regulated dephosphorylation of active MAPKs is a key component of the control of MAPK signalling cascades, and in mammals, members of the MAPK phosphatase (MKP) sub-class of dual-specificity tyrosine phosphatases have been recognized as key players for inactivating MAPKs. Five MKP homologues are found in Arabidopsis thaliana, but only limited information is available concerning their properties and biological roles. Based on initial data derived from my reverse genetics and protein interaction studies of these five potential MKPs, as well as gene function information in the literature, I chose to focus on two putative Arabidopsis MKPs, AtMKP2 and Indole-3-Butyric Acid-response 5 (IBR5). By using a combination of genetic and biochemical studies, I established that the previously uncharacterized MKP designated AtMKP2, participates in the regulation of cellular homeostasis in ozone-challenged tissue, and can influence the activation state of two MAPKs, MPK3 and MPK6. AtMKP2-suppressed plants displayed significantly prolonged MPK3 and MPK6 activation during ozone treatment, and recombinant AtMKP2 was able to dephosphorylate both phospho-MPK3 and phospho-MPK6 in vitro, providing direct evidence that AtMKP2 may target these oxidant-activated MAPKs. A mutation in IBR5, one of the five potential AtMKPs, was previously reported to confer reduced sensitivity to auxin and ABA in Arabidopsis. My protein interaction studies demonstrated that IBR5 and MPK12 are physically coupled and that the C-terminus of MPK12 is essential for its interaction with IBR5. In vitro dephosphorylation assays indicated that recombinant phosphoMPK12 is efficiently dephosphorylated by IBR5. In transgenic plants with reduced expression of the MPK12 gene, root growth is hypersensitive to exogenous auxins, consistent with the lower auxin sensitivity reported for ibr5 mutants. Taken together, my data demonstrate for the first time that both AtMKP2 and IBR5 are bona fide Arabidopsis MAPK phosphatases and that they serve as important regulators of oxidative stress and auxin signalling, respectively, in Arabidopsis.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 7 uppsatser.

Thesis (Ph.D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on March 24, 2009) Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [74]-88).