Beta integrin tyrosine phosphorylation is a conserved mechanism for regulating talin-induced integrin activation.
Integrins are large membrane-spanning receptors fundamental to cell adhesion and migration. Integrin adhesiveness for the extracellular matrix is activated by the cytoskeletal protein talin via direct binding of its phosphotyrosine-binding-like F3 domain to the cytoplasmic tail of the beta integrin subunit. The phosphotyrosine-binding domain of the signaling protein Dok1, on the other hand, has an inactivating effect on integrins, a phenomenon that is modulated by integrin tyrosine phosphorylation. Using full-length tyrosine-phosphorylated (15)N-labeled beta3, beta1A, and beta7 integrin tails and an NMR-based protein-protein interaction assay, we show that talin1 binds to the NPXY motif and the membrane-proximal portion of beta3, beta1A, and beta7 tails, and that the affinity of this interaction is decreased by integrin tyrosine phosphorylation. Dok1 only interacts weakly with unphosphorylated tails, but its affinity is greatly increased by integrin tyrosine phosphorylation. The Dok1 interaction remains restricted to the integrin NPXY region, thus phosphorylation inhibits integrin activation by increasing the affinity of beta integrin tails for a talin competitor that does not form activating membrane-proximal interactions with the integrin. Key residues governing these specificities were identified by detailed structural analysis, and talin1 was engineered to bind preferentially to phosphorylated integrins by introducing the mutation D372R. As predicted, this mutation affects talin1 localization in live cells in an integrin phosphorylation-specific manner. Together, these results indicate that tyrosine phosphorylation is a common mechanism for regulating integrin activation, despite subtle differences in how these integrins interact with their binding proteins.
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3DR, United Kingdom, California 92093. Electronic address: nick.anthis@gmail.com.
Nicholas J
NJ
Anthis
Department of Medicine, University of California, San Diego, La Jolla, California 92093.
Jacob R
JR
Haling
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3DR, United Kingdom, California 92093.
Camilla L
CL
Oxley
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3DR, United Kingdom, California 92093.
Massimiliano
M
Memo
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3DR, United Kingdom, California 92093.
Kate L
KL
Wegener
Department of Medicine, University of California, San Diego, La Jolla, California 92093.
Chinten J
CJ
Lim
Department of Medicine, University of California, San Diego, La Jolla, California 92093.
Mark H
MH
Ginsberg
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3DR, United Kingdom, California 92093. Electronic address: iain.campbell@bioch.ox.ac.uk.
Iain D
ID
Campbell
D020816:Amino Acid Motifs / Q000502:physiology; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D039641:Integrin beta Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D020125:Mutation, Missense; D019906:Nuclear Magnetic Resonance, Biomolecular; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016608:Talin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; C489561:Dok1 protein, mouse; D039641:Integrin beta Chains; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D016608:Talin; C496077:Tln1 protein, mouse; D014443:Tyrosine