Analiza funkcjonalna kompleksów kinaz map A. thaliana w warunkach stresowych
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2011-02-03T13:49:37Z
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Functional analysis of MAP kinase complexes from A. thaliana
Abstract
Rośliny posiadają bardzo ograniczone możliwości unikania stresu. Rozwinęły więc mechanizmy, umożliwiające adaptację do niekorzystnych warunków środowiska. Kinazy MAP u roślin tworzą jeden z głównych szlaków sygnalizacyjnych, który umożliwia odbiór informacji o zmianach w środowisku. Transdukcja sygnału polega na fosforylacji kolejnych kinaz MAP (MKKK, MKK oraz MPK). Stres biotyczny, abiotyczny (chłodu i osmotyczny) oraz oksydacyjny, a także hormony (ABA, kwas jasmonowy oraz etylen) aktywują przynajmniej jedną z zaledwie trzech kinaz: MPK3, MPK4 lub MPK6. Kinazy te fosforylując swoje substraty, zmieniają ich aktywność, stabilność czy lokalizację komórkową. Efektem jest ukierunkowana odpowiedź na stres, biosynteza hormonów oraz regulacja procesów rozwojowych.Celem badań było poznanie białek oddziałujących z MPK3, MPK4 i MPK6 w warunkach różnych stresów oraz podczas działania hormonów. W tym celu izolowano kompleksy białkowe kinaz, których skład poznawano na drodze spektrometrii mas. Kilka oddziaływań potwierdzono techniką BiFC. Podobnie jak oddziaływania, w różnych warunkach środowiska zmienia się fosforylacja MPK3, MPK4 i MPK6. W celu poznania bezpośrednich substratów kinaz przeprowadzono ich znakowanie za pomocą tiofosforanu, co było poprzedzone mutagenezą kinaz.
Plants as sessile organisms, exposed to a variety of environmental stresses, have developed signalling pathways leading to defensive responses. Phosphorylation/dephosphorylation networks are a common system to transduce extracellular signals. One of the most prominent components of phosphorylating modules is a mitogen-activated protein kinases (MAPKs) pathway. MAPKs generally function as a cascade, where MKKK phosphorylates and activates MKK, which activates MPK. Biotic, abiotic (cold and osmotic) and oxidative stresses as well as hormones (ABA, jasmonic acid and ethylene) activate at least one member of the MPK3, MPK4 and MPK6 trio, leading to changes in the activity, stability and cellular localization of their downstream targets. Outcome of their action are defense responses, biosynthesis of hormones, induction or inhibition of developmental programs. The aim of this study was to identify proteins interacting with MPK3, MPK4 and MPK6 in response to a variety of stresses and hormones. To this end isolations of protein complexes of kinases, formed in response to external stimuli, were carried out. Protein complexes composition was analyzed by mass spectrometry and several detected interactions were confirmed by bimolecular fluorescence complementation (BiFC). In order to detect direct substrates of kinases, thiophosphate labelling was performed. Furthermore, mass spectrometry analysis enabled to define several previously unknown phosphorylation sites of kinases. Phosphorylation of distinct amino acids was dependent on applied environmental conditions and can possibly alter kinase activity.
Plants as sessile organisms, exposed to a variety of environmental stresses, have developed signalling pathways leading to defensive responses. Phosphorylation/dephosphorylation networks are a common system to transduce extracellular signals. One of the most prominent components of phosphorylating modules is a mitogen-activated protein kinases (MAPKs) pathway. MAPKs generally function as a cascade, where MKKK phosphorylates and activates MKK, which activates MPK. Biotic, abiotic (cold and osmotic) and oxidative stresses as well as hormones (ABA, jasmonic acid and ethylene) activate at least one member of the MPK3, MPK4 and MPK6 trio, leading to changes in the activity, stability and cellular localization of their downstream targets. Outcome of their action are defense responses, biosynthesis of hormones, induction or inhibition of developmental programs. The aim of this study was to identify proteins interacting with MPK3, MPK4 and MPK6 in response to a variety of stresses and hormones. To this end isolations of protein complexes of kinases, formed in response to external stimuli, were carried out. Protein complexes composition was analyzed by mass spectrometry and several detected interactions were confirmed by bimolecular fluorescence complementation (BiFC). In order to detect direct substrates of kinases, thiophosphate labelling was performed. Furthermore, mass spectrometry analysis enabled to define several previously unknown phosphorylation sites of kinases. Phosphorylation of distinct amino acids was dependent on applied environmental conditions and can possibly alter kinase activity.
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Wydział Biologii: Instytut Biologii Molekularnej i Biotechnologii: Zakład Biotechnologii
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Keywords
Sygnalizacja, Signaling, Hormony, Hormones, Kinazy MAP, MAP kinases, Oddziaływania, Interactions