Mutations in two putative phosphorylation motifs in the tomato pollen receptor kinase LePRK2 show antagonistic effects on pollen tube length

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The tip-growing pollen tube is a useful model for studying polarized cell growth in plants. We previously characterized LePRK2, a pollen-specific receptor-like kinase from tomato (1). Here, we showed that LePRK2 is present as multiple phosphorylated isoforms in mature pollen membranes. Using compara...

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Autores principales:	Salem, T., Mazzella, A., Barberini, M.L., Wengier, D., Motillo, V., Parisi, G., Muschietti, J.
Formato:	Artículo publishedVersion
Publicado:	2011
Materias:	Amino acids Enzymes Fruits Phosphorylation Tubes (components) Alanine substitution Antagonistic effects Comparative sequence analysis Cytoplasmic juxtamembrane Phosphorylation sites Polarized cell growth Receptor-like kinase Site directed mutagenesis Plants (botany) alanine cell surface receptor protein LePRK2 serine threonine unclassified drug protein kinase vegetable protein amino acid substitution article controlled study gene overexpression growth regulation mutation nonhuman phenotype pollen tube growth priority journal protein dephosphorylation protein expression protein function protein motif protein phosphorylation protein processing sequence analysis site directed mutagenesis tomato enzymology genetics growth, development and aging metabolism pollen tube Lycopersicon esculentum Nicotiana tabacum Amino Acid Motifs Mutagenesis, Site-Directed Mutation Plant Proteins Pollen Tube Protein Kinases
Acceso en línea:	http://hdl.handle.net/20.500.12110/paper_00219258_v286_n6_p4882_Salem http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=artiaex&d=paper_00219258_v286_n6_p4882_Salem_oai
Aporte de:	Repositorio Digital de la Universidad de Buenos Aires (UBA) de Universidad de Buenos Aires

id	I28-R145-paper_00219258_v286_n6_p4882_Salem_oai
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spelling	I28-R145-paper_00219258_v286_n6_p4882_Salem_oai2020-10-19 Salem, T. Mazzella, A. Barberini, M.L. Wengier, D. Motillo, V. Parisi, G. Muschietti, J. 2011 The tip-growing pollen tube is a useful model for studying polarized cell growth in plants. We previously characterized LePRK2, a pollen-specific receptor-like kinase from tomato (1). Here, we showed that LePRK2 is present as multiple phosphorylated isoforms in mature pollen membranes. Using comparative sequence analysis and phosphorylation site prediction programs, we identified two putative phosphorylation motifs in the cytoplasmic juxtamembrane (JM) domain. Site-directed mutagenesis in these motifs, followed by transient overexpression in tobacco pollen, showed that both motifs have opposite effects in regulating pollen tube length. Relative to LePRK2-eGFP pollen tubes, alanine substitutions in residues of motif I, Ser277/Ser279/ Ser282, resulted in longer pollen tubes, but alanine substitutions in motif II, Ser304/Ser307/Thr308, resulted in shorter tubes. In contrast, phosphomimicking aspartic substitutions at these residues gave reciprocal results, that is, shorter tubes with mutations in motif I and longer tubes with mutations in motif II. We conclude that the length of pollen tubes can be negatively and positively regulated by phosphorylation of residues in motif I and II respectively. We also showed that LePRK2-eGFP significantly decreased pollen tube length and increased pollen tube tip width, relative to eGFP tubes. The kinase activity of LePRK2 was relevant for this phenotype because tubes that expressed a mutation in a lysine essential for kinase activity showed the same length and width as the eGFP control. Taken together, these results suggest that LePRK2 may have a central role in pollen tube growth through regulation of its own phosphorylation status. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Fil:Salem, T. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Mazzella, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Wengier, D. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Muschietti, J. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. application/pdf http://hdl.handle.net/20.500.12110/paper_00219258_v286_n6_p4882_Salem info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar J. Biol. Chem. 2011;286(6):4882-4891 Amino acids Enzymes Fruits Phosphorylation Tubes (components) Alanine substitution Antagonistic effects Comparative sequence analysis Cytoplasmic juxtamembrane Phosphorylation sites Polarized cell growth Receptor-like kinase Site directed mutagenesis Plants (botany) alanine cell surface receptor protein LePRK2 serine threonine unclassified drug protein kinase vegetable protein amino acid substitution article controlled study gene overexpression growth regulation mutation nonhuman phenotype pollen tube growth priority journal protein dephosphorylation protein expression protein function protein motif protein phosphorylation protein processing sequence analysis site directed mutagenesis tomato enzymology genetics growth, development and aging metabolism mutation pollen tube Lycopersicon esculentum Nicotiana tabacum Amino Acid Motifs Lycopersicon esculentum Mutagenesis, Site-Directed Mutation Plant Proteins Pollen Tube Protein Kinases Mutations in two putative phosphorylation motifs in the tomato pollen receptor kinase LePRK2 show antagonistic effects on pollen tube length info:eu-repo/semantics/article info:ar-repo/semantics/artículo info:eu-repo/semantics/publishedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=artiaex&d=paper_00219258_v286_n6_p4882_Salem_oai
institution	Universidad de Buenos Aires
institution_str	I-28
repository_str	R-145
collection	Repositorio Digital de la Universidad de Buenos Aires (UBA)
topic	Amino acids Enzymes Fruits Phosphorylation Tubes (components) Alanine substitution Antagonistic effects Comparative sequence analysis Cytoplasmic juxtamembrane Phosphorylation sites Polarized cell growth Receptor-like kinase Site directed mutagenesis Plants (botany) alanine cell surface receptor protein LePRK2 serine threonine unclassified drug protein kinase vegetable protein amino acid substitution article controlled study gene overexpression growth regulation mutation nonhuman phenotype pollen tube growth priority journal protein dephosphorylation protein expression protein function protein motif protein phosphorylation protein processing sequence analysis site directed mutagenesis tomato enzymology genetics growth, development and aging metabolism mutation pollen tube Lycopersicon esculentum Nicotiana tabacum Amino Acid Motifs Lycopersicon esculentum Mutagenesis, Site-Directed Mutation Plant Proteins Pollen Tube Protein Kinases
spellingShingle	Amino acids Enzymes Fruits Phosphorylation Tubes (components) Alanine substitution Antagonistic effects Comparative sequence analysis Cytoplasmic juxtamembrane Phosphorylation sites Polarized cell growth Receptor-like kinase Site directed mutagenesis Plants (botany) alanine cell surface receptor protein LePRK2 serine threonine unclassified drug protein kinase vegetable protein amino acid substitution article controlled study gene overexpression growth regulation mutation nonhuman phenotype pollen tube growth priority journal protein dephosphorylation protein expression protein function protein motif protein phosphorylation protein processing sequence analysis site directed mutagenesis tomato enzymology genetics growth, development and aging metabolism mutation pollen tube Lycopersicon esculentum Nicotiana tabacum Amino Acid Motifs Lycopersicon esculentum Mutagenesis, Site-Directed Mutation Plant Proteins Pollen Tube Protein Kinases Salem, T. Mazzella, A. Barberini, M.L. Wengier, D. Motillo, V. Parisi, G. Muschietti, J. Mutations in two putative phosphorylation motifs in the tomato pollen receptor kinase LePRK2 show antagonistic effects on pollen tube length
topic_facet	Amino acids Enzymes Fruits Phosphorylation Tubes (components) Alanine substitution Antagonistic effects Comparative sequence analysis Cytoplasmic juxtamembrane Phosphorylation sites Polarized cell growth Receptor-like kinase Site directed mutagenesis Plants (botany) alanine cell surface receptor protein LePRK2 serine threonine unclassified drug protein kinase vegetable protein amino acid substitution article controlled study gene overexpression growth regulation mutation nonhuman phenotype pollen tube growth priority journal protein dephosphorylation protein expression protein function protein motif protein phosphorylation protein processing sequence analysis site directed mutagenesis tomato enzymology genetics growth, development and aging metabolism mutation pollen tube Lycopersicon esculentum Nicotiana tabacum Amino Acid Motifs Lycopersicon esculentum Mutagenesis, Site-Directed Mutation Plant Proteins Pollen Tube Protein Kinases
description	The tip-growing pollen tube is a useful model for studying polarized cell growth in plants. We previously characterized LePRK2, a pollen-specific receptor-like kinase from tomato (1). Here, we showed that LePRK2 is present as multiple phosphorylated isoforms in mature pollen membranes. Using comparative sequence analysis and phosphorylation site prediction programs, we identified two putative phosphorylation motifs in the cytoplasmic juxtamembrane (JM) domain. Site-directed mutagenesis in these motifs, followed by transient overexpression in tobacco pollen, showed that both motifs have opposite effects in regulating pollen tube length. Relative to LePRK2-eGFP pollen tubes, alanine substitutions in residues of motif I, Ser277/Ser279/ Ser282, resulted in longer pollen tubes, but alanine substitutions in motif II, Ser304/Ser307/Thr308, resulted in shorter tubes. In contrast, phosphomimicking aspartic substitutions at these residues gave reciprocal results, that is, shorter tubes with mutations in motif I and longer tubes with mutations in motif II. We conclude that the length of pollen tubes can be negatively and positively regulated by phosphorylation of residues in motif I and II respectively. We also showed that LePRK2-eGFP significantly decreased pollen tube length and increased pollen tube tip width, relative to eGFP tubes. The kinase activity of LePRK2 was relevant for this phenotype because tubes that expressed a mutation in a lysine essential for kinase activity showed the same length and width as the eGFP control. Taken together, these results suggest that LePRK2 may have a central role in pollen tube growth through regulation of its own phosphorylation status. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
format	Artículo Artículo publishedVersion
author	Salem, T. Mazzella, A. Barberini, M.L. Wengier, D. Motillo, V. Parisi, G. Muschietti, J.
author_facet	Salem, T. Mazzella, A. Barberini, M.L. Wengier, D. Motillo, V. Parisi, G. Muschietti, J.
author_sort	Salem, T.
title	Mutations in two putative phosphorylation motifs in the tomato pollen receptor kinase LePRK2 show antagonistic effects on pollen tube length
title_short	Mutations in two putative phosphorylation motifs in the tomato pollen receptor kinase LePRK2 show antagonistic effects on pollen tube length
title_full	Mutations in two putative phosphorylation motifs in the tomato pollen receptor kinase LePRK2 show antagonistic effects on pollen tube length
title_fullStr	Mutations in two putative phosphorylation motifs in the tomato pollen receptor kinase LePRK2 show antagonistic effects on pollen tube length
title_full_unstemmed	Mutations in two putative phosphorylation motifs in the tomato pollen receptor kinase LePRK2 show antagonistic effects on pollen tube length
title_sort	mutations in two putative phosphorylation motifs in the tomato pollen receptor kinase leprk2 show antagonistic effects on pollen tube length
publishDate	2011
url	http://hdl.handle.net/20.500.12110/paper_00219258_v286_n6_p4882_Salem http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=artiaex&d=paper_00219258_v286_n6_p4882_Salem_oai
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Mutations in two putative phosphorylation motifs in the tomato pollen receptor kinase LePRK2 show antagonistic effects on pollen tube length

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