Search

Ca2+ release is tightly sequestered in eukaryotic cells to enable fine spatio-temporal control of signalling but how Ca2+ release from the endoplasmic reticulum (ER) is linked to cell adhesions is not defined. We examined the spatial restriction of Ca2+ release through the inositol 1,4,5-triphosphate receptor 1 (IP3R1) in response to interleukin-1 (IL-1) and the functions of the adhesion-associated proteins, focal adhesion kinase (FAK) and protein tyrosine phosphatase-α (PTPα). In cultured fibroblasts IL-1 treatment promoted co-localization of PTPα and FAK with the ER and increased association of IP3R1 with PTPα and FAK at focal adhesions (FAs). GST pull-down assays of purified proteins demonstrated that PTPα and FAK directly interacted with IP3R1. These interactions depended on the focal adhesion-targeting (FAT) and band4.1-ezrin-radixin-moesin (FERM) domains of FAK. PTPα was required for the association of IP3R1 with Src, which mediated IP3R1 phosphorylation and consequently ER Ca2+ release. Collectively, these data indicate that PTPα and FAK, which are enriched in FAs, interact with IP3R1 at adjacent ER sites to spatially sequester IL-1-induced Ca2+ signalling. [ABSTRACT FROM AUTHOR]

IL (interleukin)-1 signalling in anchorage-dependent cells involves focal-adhesion-restricted and Ca2+ -dependent Ras and ERK (extracellular-signal-regulated kinase) activation that leads to MMP (matrix metalloproteinase) release and extracellular matrix remodelling. Ras activity is regulated, in part, by the Ca2+ - responsive Ras GRFs (guanine-nucleotide-releasing factors) 1 and 2, but the mechanisms that link and localize IL-1-induced Ca2+ signalling to focal adhesions are not defined. In the present study we characterized the role of Ras-GRF1/2 in Ca2+ and Ras?ERK signalling after IL-1 stimulation. By immunoprecipitation we found that Ras-GRF1/2 associates with PLC? 1 (phospholipase C? 1). This association enables PLC? 1 recruitment to focal adhesions and is required for Ras signalling, ERK activation and MMP-3 release downstream of IL-1 stimulation. Depletion of PLC? 1 by siRNA (small interfering RNA) abolished IL-1-induced Ras activation and MMP-3 expression. Buffering of cytosolic Ca2+ reduced Ras interactions with Ras-GRF1/2 and blocked MMP-3 release. The results of the present study showthat, in addition to their functions as Ras-exchange factors, Ras-GRF1 and -GRF2 may act as adaptors that bind PLC? 1 and restrict Ca2+ signalling to the vicinity of focal adhesions, indicating a new role for these GRFs that is required for IL-1 induction of the Ras?ERK pathway and MMP-3 expression. [ABSTRACT FROM AUTHOR]