RNAi Library Screening Reveals Gβ 1 , Casein Kinase 2α and ICAP-1 as
Novel Regulators of LFA-1-mediated T-cell Polarity and Migration
Abstract
The α Lβ 2 integrin LFA-1 plays a key
role in T-cell adhesion to the endothelial vasculature and migration
into both secondary lymphoid organs and peripheral tissues via
interactions with its target protein ICAM-1. Although members of the
chemokine family of G-protein coupled receptors and heterotrimeric G
proteins are known to influence LFA-1 activation and ICAM binding, the
role of these receptors and their downstream effectors has not been
fully characterised. In addition, a number of kinases are activated in
response to LFA-1/ICAM-1 signalling in T-cells but the extent to which
the kinome influences T-cell migration is not fully understood. In this
study we screened two RNAi libraries targeting G protein-coupled
receptors (GPCR)/GPCR-associated proteins and kinases in a HuT-78 T-cell
line model of LFA-1-stimulated T cell migration. Based on staining of
the actin cytoskeleton, multiple parameters to measure cell morphology
were used to assess the contribution of 1109 genes to LFA-1-mediated T
cell polarity and migration. These RNAi screens identified a number of
both novel and previously identified genes that either increased or
decreased the polarity and migratory capacity of these cells. Following
multiparametric analysis, hierarchical clustering and pathway analysis,
three of these genes were characterised in further detail using primary
human T-cells, revealing novel roles for the heterotrimeric G protein
subunit Gβ1 and casein kinase 2 in LFA-1-mediated T-cell polarity and
migration in-vitro. Our studies also highlighted a new role for ICAP-1,
an adaptor protein previously described to be associated with β1
integrins, in β2 integrin LFA-1-directed migration in T-cells. Knockdown
of ICAP-1 expression in primary T cells revealed a role in cell
polarity, cell velocity and transmigration towards SDF-1 for this
adaptor protein. This study therefore uncovers new roles for
GPCR/GPCR-associated proteins and kinases in T-cell migration and
provides potential novel targets for modulation of the T-cell immune
response.