Analysis of dynamic tyrosine phosphoproteome in LFA‐1 triggered migrating t‐cells

Abstract

The ordered, directional migration of T-lymphocytes is a key process during immune surveillance and response. This requires cell adhesion to the high endothelial venules or to the extracellular matrix by a series of surface receptor/ligand interactions involving adhesion molecules of the integrin family including lymphocyte function associated molecule-1 (LFA-1) and intercellular adhesion molecules (ICAMs). Reversible protein phosphorylation is emerging as a key player in the regulation of biological functions with tyrosine phosphorylation playing a crucial role in signal transduction. Thus, the study of this type of post-translational modification at the proteomic level has great biological significance. In this work, phospho-enriched cell lysates from LFA-1-triggered migrating human T-cells were subjected to immunoaffinity purification of tyrosine phosphorylated proteins, mass spectrometric and bioinformatic analysis.In addition to the identification of several well-documented proteins, the analysis suggested involvement of a number of new and novel proteins in LFA-1 induced T-cell migration. This dataset expands the list of the signalling components of the LFA-1 induced phosphotyrosine protein complexes in migrating T-cells that will be extremely useful in the study of their specific roles within LFA-1 associated signalling pathways. Identification of proteins previously not reported in the context of LFA-1 stimulated signal transduction may provide new insights into understanding the LFA-1 signalling networks and aid in the search for new potential therapeutic targets.

LFA-1/ICAM-1 Ligation in Human T Cells Promotes Th1 Polarization through a GSK3β Signaling–Dependent Notch Pathway

“…The protein content of the cell lysates was determined by Bradford or BCA assay. The immunoprecipitation followed by SDS-PAGE of the cellular lysates or immunoprecipitates and subsequent Western immunoblotting using indicated Abs were performed as described (22). The immunoreactive bands were visualized using the LumiGLO chemiluminescent detection system (Cell Signaling Technology) and subsequent exposure to CL-XPosure light-sensitive film (Thermo Scientific).…”

Phosphorylation of Rab5a Protein by Protein Kinase Cϵ Is Crucial for T-cell Migration

“…The human T-cell line HuT-78 was obtained from the American Type Culture collection (ATCC) and cultured as described (27)(28)(29)(30). Motility-inducing anti-LFA-1 (clone SPV-L7) was from Monosan (Cambridge Biosciences).…”

“…The protein content of the cell lysates was determined by the Bradford or BCA assay. The immunoprecipitation was performed as described previously (28,33). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the cellular lysates or immunoprecipitates and subsequent Western immunoblotting using indicated antibodies were performed as described (28).…”

“…The immunoprecipitation was performed as described previously (28,33). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the cellular lysates or immunoprecipitates and subsequent Western immunoblotting using indicated antibodies were performed as described (28). Where indicated, affinity-purified or crude anti-phospho Rab5a(Thr-7) antiserum (1:2000 dilution in blocking buffer) was preincubated with 20 g/ml non-phosphorylated Thr-7 peptide for 1 h at room temperature before incubation on the membranes.…”

Conjugated Linoleic Acid Targets β2 Integrin Expression To Suppress Monocyte Adhesion

“…Leukocytes binding to ICAM-1 develop a polarized promigratory phenotype with an actin-rich leading edge and a trailing uropod (30). To examine whether CLA treatment could also prevent polarization of monocytes, we pretreated monocytes with CLA as before and incubated them on ICAM-1-coated plates.…”