Genome-wide analysis of Arabidopsis thaliana with tyrosine kinase motif from animals predicted th... more Genome-wide analysis of Arabidopsis thaliana with tyrosine kinase motif from animals predicted that tyrosine phosphorylation could be brought about only by dual-speciWcity protein kinases in plants. However, their regulation is poorly understood. In the present study, we have investigated the role of serines required for the activity of Arabidopsis thaliana serine/threonine/tyrosine protein kinase (AtSTYPK). There are eight serines in the kinase catalytic domain. The role of each serine residue was studied individually by substituting them with alanine. Serines at positions 215, 259, 269 and 315 are required for the kinase activity both in terms of auto and substrate phosphorylations of myelin basic protein. The mutant S265A showed slight increase in auto and substrate phosphorylations. Other serines at positions 165, 181 and 360 did not show any change in the phosphorylation status as compared to wild-type. In conclusion, these results suggest the importance of serine residues required for dual-speciWcity protein kinase activity.
Tyrosine phosphorylation in plants could be performed only by dual-speciWcity kinases. Arabidopsi... more Tyrosine phosphorylation in plants could be performed only by dual-speciWcity kinases. Arabidopsis thaliana dual-speciWcity protein kinase (AtSTYPK) exhibited strong preference for manganese over magnesium for its kinase activity. The kinase autophosphorylated on serine, threonine and tyrosine residues and phosphorylated myelin basic protein on threonine and tyrosine residues. The AtSTYPK harbors manganese dependent serine/threonine kinase domain, COG3642. His 248 and Ser 265 on COG3642 are conserved in AtSTYPK and the site-directed mutant, H248A showed loss of serine/threonine kinase activity. The protein kinase activity was abolished when Thr 208 in the TEY motif and Thr 293 of the activation loop were converted to alanine. The conversion of Thr 284 in the activation loop to alanine resulted in an increased phosphorylation. This study reports the Wrst identiWcation of a manganese dependent dual-speciWcity kinase and the importance of Thr 208 , Thr 284 , and Thr 293 residues in the regulation of kinase activity.
Protein tyrosine phosphorylation is carried out by dual-specificity kinases in plants. Peanut dua... more Protein tyrosine phosphorylation is carried out by dual-specificity kinases in plants. Peanut dual-specificity kinase has been shown to be regulated by tyrosine phosphorylation. However, the role of threonine residues in the regulation of peanut serine/threonine/tyrosine (STY) protein kinase is not yet documented. In the present study, we have investigated the role of threonine residues in the regulation of peanut STY protein kinase activity. The four threonine residues in the kinase activation loop and Thr 211 in the threonine-glutamate-tyrosine (TEY) motif were mutated to alanine to study their role in the regulation of kinase activity. The protein kinase activity was abolished when Thr 211 of TEY motif and Thr 296 of activation loop were converted to alanine, suggesting that they positively regulate the kinase activity. The ability of T211A and T296A to phosphorylate histone H1 was also reduced drastically. The other mutants T287A, T291A and T294A did not show any change in their ability to autophosphorylate or phosphorylate histone H1 when compared to wild-type peanut STY protein kinase. Data presented here suggests the importance of threonine residues in the regulation of peanut STY protein kinase activity and emphasizes the complexity of regulation of dualspecificity protein kinases in plants. #
Protein tyrosine phosphorylation plays an important role in cell growth, development and oncogene... more Protein tyrosine phosphorylation plays an important role in cell growth, development and oncogenesis. No classical protein tyrosine kinase has hitherto been cloned from plants. Does protein tyrosine kinase exist in plants? To address this, we have performed a genomic survey of protein tyrosine kinase motifs in plants using the delineated tyrosine phosphorylation motifs from the animal system. The Arabidopsis thaliana genome encodes 57 different protein kinases that have tyrosine kinase motifs. Animal non-receptor tyrosine kinases, SRC, ABL, LYN, FES, SEK, KIN and RAS have structural relationship with putative plant tyrosine kinases. In an extended analysis, animal receptor and non-receptor kinases, Raf and Ras kinases, mixed lineage kinases and plant serine/threonine/tyrosine (STY) protein kinases, form a well-supported group sharing a common origin within the superfamily of STY kinases. We report that plants lack bona fide tyrosine kinases, which raise an intriguing possibility that tyrosine phosphorylation is carried out by dual-specificity STY protein kinases in plants. The distribution pattern of STY protein kinase families on Arabidopsis chromosomes indicates that this gene family is partly a consequence of duplication and reshuffling of the Arabidopsis genome and of the generation of tandem repeats. Genome-wide analysis is supported by the functional expression and characterization of At2g24360 and phosphoproteomics of Arabidopsis. Evidence for tyrosine phosphorylated proteins is provided by alkaline hydrolysis, anti-phosphotyrosine immunoblotting, phosphoamino acid analysis and peptide mass fingerprinting. These results report the first comprehensive survey of genome-wide and tyrosine phosphoproteome analysis of plant STY protein kinases.
Genome-wide analysis of Arabidopsis thaliana with tyrosine kinase motif from animals predicted th... more Genome-wide analysis of Arabidopsis thaliana with tyrosine kinase motif from animals predicted that tyrosine phosphorylation could be brought about only by dual-speciWcity protein kinases in plants. However, their regulation is poorly understood. In the present study, we have investigated the role of serines required for the activity of Arabidopsis thaliana serine/threonine/tyrosine protein kinase (AtSTYPK). There are eight serines in the kinase catalytic domain. The role of each serine residue was studied individually by substituting them with alanine. Serines at positions 215, 259, 269 and 315 are required for the kinase activity both in terms of auto and substrate phosphorylations of myelin basic protein. The mutant S265A showed slight increase in auto and substrate phosphorylations. Other serines at positions 165, 181 and 360 did not show any change in the phosphorylation status as compared to wild-type. In conclusion, these results suggest the importance of serine residues required for dual-speciWcity protein kinase activity.
Tyrosine phosphorylation in plants could be performed only by dual-speciWcity kinases. Arabidopsi... more Tyrosine phosphorylation in plants could be performed only by dual-speciWcity kinases. Arabidopsis thaliana dual-speciWcity protein kinase (AtSTYPK) exhibited strong preference for manganese over magnesium for its kinase activity. The kinase autophosphorylated on serine, threonine and tyrosine residues and phosphorylated myelin basic protein on threonine and tyrosine residues. The AtSTYPK harbors manganese dependent serine/threonine kinase domain, COG3642. His 248 and Ser 265 on COG3642 are conserved in AtSTYPK and the site-directed mutant, H248A showed loss of serine/threonine kinase activity. The protein kinase activity was abolished when Thr 208 in the TEY motif and Thr 293 of the activation loop were converted to alanine. The conversion of Thr 284 in the activation loop to alanine resulted in an increased phosphorylation. This study reports the Wrst identiWcation of a manganese dependent dual-speciWcity kinase and the importance of Thr 208 , Thr 284 , and Thr 293 residues in the regulation of kinase activity.
Protein tyrosine phosphorylation is carried out by dual-specificity kinases in plants. Peanut dua... more Protein tyrosine phosphorylation is carried out by dual-specificity kinases in plants. Peanut dual-specificity kinase has been shown to be regulated by tyrosine phosphorylation. However, the role of threonine residues in the regulation of peanut serine/threonine/tyrosine (STY) protein kinase is not yet documented. In the present study, we have investigated the role of threonine residues in the regulation of peanut STY protein kinase activity. The four threonine residues in the kinase activation loop and Thr 211 in the threonine-glutamate-tyrosine (TEY) motif were mutated to alanine to study their role in the regulation of kinase activity. The protein kinase activity was abolished when Thr 211 of TEY motif and Thr 296 of activation loop were converted to alanine, suggesting that they positively regulate the kinase activity. The ability of T211A and T296A to phosphorylate histone H1 was also reduced drastically. The other mutants T287A, T291A and T294A did not show any change in their ability to autophosphorylate or phosphorylate histone H1 when compared to wild-type peanut STY protein kinase. Data presented here suggests the importance of threonine residues in the regulation of peanut STY protein kinase activity and emphasizes the complexity of regulation of dualspecificity protein kinases in plants. #
Protein tyrosine phosphorylation plays an important role in cell growth, development and oncogene... more Protein tyrosine phosphorylation plays an important role in cell growth, development and oncogenesis. No classical protein tyrosine kinase has hitherto been cloned from plants. Does protein tyrosine kinase exist in plants? To address this, we have performed a genomic survey of protein tyrosine kinase motifs in plants using the delineated tyrosine phosphorylation motifs from the animal system. The Arabidopsis thaliana genome encodes 57 different protein kinases that have tyrosine kinase motifs. Animal non-receptor tyrosine kinases, SRC, ABL, LYN, FES, SEK, KIN and RAS have structural relationship with putative plant tyrosine kinases. In an extended analysis, animal receptor and non-receptor kinases, Raf and Ras kinases, mixed lineage kinases and plant serine/threonine/tyrosine (STY) protein kinases, form a well-supported group sharing a common origin within the superfamily of STY kinases. We report that plants lack bona fide tyrosine kinases, which raise an intriguing possibility that tyrosine phosphorylation is carried out by dual-specificity STY protein kinases in plants. The distribution pattern of STY protein kinase families on Arabidopsis chromosomes indicates that this gene family is partly a consequence of duplication and reshuffling of the Arabidopsis genome and of the generation of tandem repeats. Genome-wide analysis is supported by the functional expression and characterization of At2g24360 and phosphoproteomics of Arabidopsis. Evidence for tyrosine phosphorylated proteins is provided by alkaline hydrolysis, anti-phosphotyrosine immunoblotting, phosphoamino acid analysis and peptide mass fingerprinting. These results report the first comprehensive survey of genome-wide and tyrosine phosphoproteome analysis of plant STY protein kinases.
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Papers by Mamatha Reddy