Beta2 integrin phosphorylation on Thr758 acts as a molecular switch to regulate 14-3-3 and filamin binding.
Leukocyte integrins of the beta2 family are essential for immune cell-cell adhesion. In activated cells, beta2 integrins are phosphorylated on the cytoplasmic Thr758, leading to 14-3-3 protein recruitment to the beta2 integrin. The mutation of this phosphorylation site impairs cell adhesion, actin reorganization, and cell spreading. Thr758 is contained in a Thr triplet of beta2 that also mediates binding to filamin. Here, we investigated the binding of filamin, talin, and 14-3-3 proteins to phosphorylated and unphosphorylated beta2 integrins by biochemical methods and x-ray crystallography. 14-3-3 proteins bound only to the phosphorylated integrin cytoplasmic peptide, with a high affinity (K(d), 261 nM), whereas filamin bound only the unphosphorylated integrin cytoplasmic peptide (K(d), 0.5 mM). Phosphorylation did not regulate talin binding to beta2 directly, but 14-3-3 was able to outcompete talin for the binding to phosphorylated beta2 integrin. X-ray crystallographic data clearly explained how phosphorylation eliminated filamin binding and induced 14-3-3 protein binding. Filamin knockdown in T cells led to an increase in stimulated cell adhesion to ICAM-1-coated surfaces. Our results suggest that the phosphorylation of beta2 integrins on Thr758 acts as a molecular switch to inhibit filamin binding and allow 14-3-3 protein binding to the integrin cytoplasmic domain, thereby modulating T-cell adhesion.
Department of Biological and Environmental Science and Nanoscience Center, University of Jyväskylä, Jyväskylä, Finland.
Heikki
H
Takala
Elisa
E
Nurminen
Susanna M
SM
Nurmi
Maria
M
Aatonen
Tomas
T
Strandin
Maarit
M
Takatalo
Tiila
T
Kiema
Carl G
CG
Gahmberg
Jari
J
Ylänne
Susanna C
SC
Fagerholm
D048948:14-3-3 Proteins / Q000737:chemistry / Q000378:metabolism*; D019943:Amino Acid Substitution; D001665:Binding Sites; D018821:CD18 Antigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002448:Cell Adhesion; D003285:Contractile Proteins / Q000737:chemistry / Q000378:metabolism*; D064448:Filamins; D006801:Humans; D066298:In Vitro Techniques; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D019169:Jurkat Cells; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D046912:Multiprotein Complexes; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D013601:T-Lymphocytes / Q000378:metabolism; D016608:Talin / Q000378:metabolism; D013912:Threonine / Q000737:chemistry
D048948:14-3-3 Proteins; D018821:CD18 Antigens; D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D016608:Talin; D018799:Intercellular Adhesion Molecule-1; D013912:Threonine