Threonine eliminylation by bacterial phosphothreonine lyases rapidly causes cross-linking of mitogen-activated protein kinase (MAPK) in live cells.
Old long-lived proteins contain dehydroalanine (Dha) and dehydrobutyrine (Dhb), two amino acids engendered by dehydration of serines and threonines, respectively. Although these residues have a suspected role in protein cross-linking and aggregation, their direct implication has yet to be determined. Here, we have taken advantage of the ability of the enteropathogenShigellato convert the phosphothreonine residue of the pT-X-pY consensus sequence of ERK and p38 into Dhb and followed the impact of dehydration on the fate of the two MAPKs. To that end, we have generated the first antibodies recognizing Dhb-modified proteins and allowing tracing them as they form. We showed that Dhb modifications accumulate in a long-lasting mannerin Shigella-infected cells, causing subsequent formation of covalent cross-links of MAPKs. Moreover, the Dhb signal correlates precisely with the activation of theShigellatype III secretion apparatus, thus evidencing injectisome activity. This observation is the first to document a causal link between Dhb formation and protein cross-linking in live cells. Detection of eliminylation is a new avenue to phosphoproteome regulation in eukaryotes that will be instrumental for the development of type III secretion inhibitors.
From the Team genomic plasticity and infection, Department of Immunology, Infectiology and Hematology, Institut Necker Enfants Malades, INSERM U1151, CNRS UMR 8253, 75993 Paris CEDEX 14, France.|the Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur UPMC, 75724 Paris, France.
Benoit M
BM
Meijer
Institut Pasteur, Department of Biologie du Développement et Cellules Souches, Unité de Régulation Epigénétique, 75724 Paris CEDEX 15, France.|UMR3738 CNRS, 75732 Paris CEDEX 15, France.
Suk Min
SM
Jang
the Proteomic Platform Necker, PPN-3P5, Structure Fédérative de Recherche SFR Necker US24, 75015 Paris, France.
Ida C
IC
Guerrera
the Proteomic Platform Necker, PPN-3P5, Structure Fédérative de Recherche SFR Necker US24, 75015 Paris, France.
Cerina
C
Chhuon
the Proteomic Platform Necker, PPN-3P5, Structure Fédérative de Recherche SFR Necker US24, 75015 Paris, France.|the CPN Proteomics Facility-3P5, Center of Psychiatry and Neuroscience, UMR INSERM 894, 75014 Paris, France.
Joanna
J
Lipecka
From the Team genomic plasticity and infection, Department of Immunology, Infectiology and Hematology, Institut Necker Enfants Malades, INSERM U1151, CNRS UMR 8253, 75993 Paris CEDEX 14, France.
Caroline
C
Reisacher
the Unité de Chimie des Biomolécules, Institut Pasteur, 75015 Paris, France, and.
Françoise
F
Baleux
the Unité de Pathogénie Microbienne Moléculaire, Unité INSERM U1202, Institut Pasteur, 75015 Paris, France.
Philippe J
PJ
Sansonetti
Institut Pasteur, Department of Biologie du Développement et Cellules Souches, Unité de Régulation Epigénétique, 75724 Paris CEDEX 15, France.|UMR3738 CNRS, 75732 Paris CEDEX 15, France.
Christian
C
Muchardt
From the Team genomic plasticity and infection, Department of Immunology, Infectiology and Hematology, Institut Necker Enfants Malades, INSERM U1151, CNRS UMR 8253, 75993 Paris CEDEX 14, France, laurence.arbibe@inserm.fr.|Université Paris Descartes,75270 Paris CEDEX 06, France.
Laurence
L
Arbibe
D000409:Alanine / Q000031:analogs & derivatives / Q000737:chemistry; D000613:Aminobutyrates / Q000737:chemistry; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism*; D018938:Caco-2 Cells; D019757:Carbon-Oxygen Lyases / Q000378:metabolism*; D002460:Cell Line; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D020935:MAP Kinase Signaling System; D051379:Mice; D011485:Protein Binding; D040901:Proteomics; D012760:Shigella / Q000201:enzymology*; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry*; D000069296:Type III Secretion Systems; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D000613:Aminobutyrates; D000906:Antibodies; D001426:Bacterial Proteins; D000069296:Type III Secretion Systems; C088694:dehydrobutyrine; D013912:Threonine; C015102:dehydroalanine; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D019757:Carbon-Oxygen Lyases; D000409:Alanine
Shigella; aging; bacterial protein phosphatase; dehydroalanine; dehydrobutyrine; mitogen-activated protein kinase (MAPK); phosphoproteomics; protein aggregation; protein cross-link; type III secretion system (T3SS)