Alexey Shiryaev1, Sergiy Kostenko, Gianina Dumitriu, Ugo Moens. 1. Alexey Shiryaev, Sergiy Kostenko, Gianina Dumitriu, Ugo Moens, Department of Medical Biology, Faculty of Health Sciences, University of Tromsø, N-9037 Tromsø, Norway.
Abstract
AIM: To identify novel substrates for the mitogen-activated protein kinase-activated protein kinase 5 (MK5). METHODS: Yeast two-hybrid screening with MK5 as bait was used to identify novel possible interaction partners. The binding of putative partner was further examined by glutathione S-transferase (GST) pull-down, co-immunoprecipitation and fluorescence resonance energy transfer (FRET) analysis. In vitro kinase and peptide array assays were used to map MK5 phosphoacceptor sites on the new partner. Confocal microscopy was performed to study the subcellular localization of MK5 and its partners. RESULTS: Septin 8 was identified as a novel interaction partner for MK5 by yeast two-hybrid screening. This interaction was confirmed by GST pull-down, co-immunoprecipitation and FRET analysis. Septin 5, which can form a complex with septin 8, did not interact with MK5. Serine residues 242 and 271 on septin 8 were identified as in vitro MK5 phosphorylation sites. MK5 and septin 8 co-localized in the perinuclear area and in cell protrusions. Moreover, both proteins co-localized with vesicle marker synaptophysin. CONCLUSION: Septin 8 is a bona fide interaction partner and in vitro substrate for MK5. This interaction may be implicated in vesicle trafficking.
AIM: To identify novel substrates for the mitogen-activated protein kinase-activated protein kinase 5 (MK5). METHODS: Yeast two-hybrid screening with MK5 as bait was used to identify novel possible interaction partners. The binding of putative partner was further examined by glutathione S-transferase (GST) pull-down, co-immunoprecipitation and fluorescence resonance energy transfer (FRET) analysis. In vitro kinase and peptide array assays were used to map MK5 phosphoacceptor sites on the new partner. Confocal microscopy was performed to study the subcellular localization of MK5 and its partners. RESULTS:Septin 8 was identified as a novel interaction partner for MK5 by yeast two-hybrid screening. This interaction was confirmed by GST pull-down, co-immunoprecipitation and FRET analysis. Septin 5, which can form a complex with septin 8, did not interact with MK5. Serine residues 242 and 271 on septin 8 were identified as in vitro MK5 phosphorylation sites. MK5 and septin 8 co-localized in the perinuclear area and in cell protrusions. Moreover, both proteins co-localized with vesicle marker synaptophysin. CONCLUSION:Septin 8 is a bona fide interaction partner and in vitro substrate for MK5. This interaction may be implicated in vesicle trafficking.
Entities:
Keywords:
Fluorescence resonance energy transfer; Mitogen-activated protein kinase-activated protein kinase-5; Phosphorylation; Septin; Synaptophysin
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