Determinants of phosphatidylinositol-4-phosphate 5-kinase type Iγ90 uropod location in T-lymphocytes and its role in uropod formation

Abstract

We have previously identified phosphatidylinositol-4-phosphate 5-kinase type I (PIPKI)γ90 as a T cell uropod component. However, the molecular determinants and functional consequences of its localization remain unknown. In this report, we seek to better understand the mechanisms involved in PIPKIγ90 uropod targeting and the role that PIPKIγ90 plays in T cell uropod formation. During T cell activation, PIPKIγ90 cocaps with the membrane microdomain-associated proteins flotillin-1 and -2 and accumulates in the uropod. We report that the C-terminal 26 amino acid extension of PIPKIγ90 is required for its localization to the uropod. We further use T cells from PIPKIγ90−/− mice and human T cells expressing a kinase-dead PIPKIγ90 mutant to examine the role of PIPKIγ90 in a T cell uropod formation. We find that PIPKIγ90 deficient T cells have elongated uropods on ICAM-1. Moreover, in human T cells overexpression of PIPKIγ87, a naturally occurring isoform lacking the last 26 amino acids, suppresses uropod formation and impairs capping of uropod proteins such as flotillins. Transfection of human T cells with a dominant-negative mutant of flotillin-2 in turn attenuates capping of PIPKIγ90. Our data contribute to the understanding of the molecular mechanisms that regulate T cell uropod formation.

Analysis of close associations of uropod-associated proteins in human T-cells using the proximity ligation assay

“…GFP-tagged PIPKIγ90 accumulates in the uropod of murine T-cells (Lokuta et al, 2007; Mathis et al, 2013). We expressed an N-terminally HA-tagged PIPKIγ90 construct in human T-cells and observed colocalization of this construct with P-ERM in the uropod (Fig.…”

“…The uropod is a plasma membrane protrusion located in the rear of migrating leukocytes that contains specific organelles along with cytoskeletal, adhesion and signaling proteins such as activated phosphorylated ezrin/radixin/moesin proteins (P-ERM), the adhesion receptor P-selectin glycoprotein-1 (PSGL-1) and phosphatidylinositol-4-phosphate 5-kinase type I (PIPKI) γ90 (Lokuta et al, 2007; Sánchez-Madrid & Serrador, 2009; Mathis et al, 2013). Flotillins, membrane microdomain scaffolding proteins, are also enriched in leukocyte uropods and are involved in uropod formation (Ludwig et al, 2010; Rossy et al, 2009; Affentranger et al, 2011; Baumann, Affentranger & Niggli, 2012).…”

“…Transfection of human freshly isolated T-cells with a dominant-negative mutant of flotillin-2 impaired cell polarization and uropod capping of endogenous flotillin-1, PSGL-1 and GFP-tagged PIPKIγ90, indicating a functional role of flotillins in structuring the uropod (Affentranger et al, 2011; Mathis et al, 2013). Moreover, expression of constitutively active ezrin in freshly isolated T-cells induced capping of flotillins and PSGL-1, and that of a dominant-negative ezrin mutant impaired flotillin and PSGL-1 capping, suggesting a scaffolding role also for P-ERM (Martinelli et al, 2013).…”

“…Moreover, expression of constitutively active ezrin in freshly isolated T-cells induced capping of flotillins and PSGL-1, and that of a dominant-negative ezrin mutant impaired flotillin and PSGL-1 capping, suggesting a scaffolding role also for P-ERM (Martinelli et al, 2013). Similarly overexpression of PIPKIγ87, a naturally occurring isoform lacking the last 26 amino acids, which does not locate to the uropod, suppresses T-cell uropod formation and impairs capping of uropod proteins such as flotillins (Mathis et al, 2013). …”

PIP kinases define PI4,5P2 signaling specificity by association with effectors

“…PIPKIγi2 also accumulates at the leading edges of migrating breast cancer cells [38]. In chemotacting neutrophils and T lymphocytes, PIPKIγi2 targets to uropods at the rear of cells [51, 52]. PIPKIβ also targets to uropods of chemotacting neutrophils [53].…”