Abstract
Interleukin-1 (IL-1) signaling in fibroblasts is mediated through focal adhesions, organelles that are enriched with adaptor and cytoskeletal proteins that regulate signal transduction. We examined interactions of the focal adhesion kinase (FAK) with protein-tyrosine phosphatase-α (PTP-α) in IL-1 signaling. In wild type and FAK knock-out mouse embryonic fibroblasts, we found by immunoblotting, immunoprecipitation, immunostaining, and gene silencing that FAK is required for IL-1-mediated sequestration of PTPα to focal adhesions. Immunoprecipitation and pulldown assays of purified proteins demonstrated a direct interaction between FAK and PTPα, which was dependent on the FAT domain of FAK and by an intact membrane-proximal phosphatase domain of PTPα. Recruitment of PTPα to focal adhesions, IL-1-induced Ca(2+) release from the endoplasmic reticulum, ERK activation, and IL-6, MMP-3, and MMP-9 expression were all blocked in FAK knock-out fibroblasts. These processes were restored in FAK knock-out cells transfected with wild type FAK, FAT domain, and FRNK. Our data indicate that IL-1-induced signaling through focal adhesions involves interactions between the FAT domain of FAK and PTPα.
Highlights
Interleukin-1 signaling in fibroblasts is mediated through focal adhesions, which are enriched with tyrosine kinases and phosphatases
Accumulation of protein-tyrosine kinases and phosphatases (PTPs)␣ in FAs Requires focal adhesion kinase (FAK)—Focal adhesions are enriched with tyrosine phosphatases that may regulate signaling through FAs [34]
To assess the fibroblasts used in the current experiments and to confirm key phenotypic features, we determined whether IL-1 affects the association of PTP␣ with FA- and ERassociated proteins in FAKϩ/ϩ mouse embryonic fibroblasts (MEFs)
Summary
Interleukin-1 signaling in fibroblasts is mediated through focal adhesions, which are enriched with tyrosine kinases and phosphatases. Spatial co-localization of interacting signaling molecules provides a pivotal regulatory locus for signal transduction In adherent cells such as fibroblasts, the formation of FAs is required for IL-1-induced signaling cascades leading to tyrosine phosphorylation and activation of the focal adhesion kinase [11], Ca2ϩ release from the endoplasmic reticulum (ER) [12, 13], and activation of ERK [14, 15], which result in regulating the expression of MMPs [6, 16]. Phospholipase C␥ catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate generating a secondary messenger, inositol 1,4,5-trisphosphate, which mobilizes Ca2ϩ, whereas Shc and Grb serve as adaptors for signal transduction by the ERK pathway These processes indicate that FAK may be important in coupling components of Ca2ϩ and ERK signaling downstream of the IL-1 receptor by regulating tyrosine phosphorylation status of other signaling molecules and by interacting with other adaptor proteins. We examined the functional importance of FAK and three of its domains (band4.1-ezrinradixin-moesin (FERM) homology domain, kinase domain, and focal adhesion-targeting (FAT) domain) in influencing the interactions between FAK and PTP␣, as well as the impact on IL-1-induced signaling that leads to Ca2ϩ release, ERK activation, and IL-6, MMP-3, and MMP-9 expression
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