| Abstrakt: |
The brain-specific 42-kDa protein, p42IP4, contains a N-terminal zinc finger (ZF) motif and a tandem of two pleckstrin homology (PH) domains. p42IP4 binds in vitro the second messengers phosphatidylinositol(3,4,5)trisphosphate (PtdIns(3,4,5)P3) and inositol(1,3,4,5)tetrakisphosphate (Ins(1,3,4,5)P4). We observed by confocal microscopy in live HEK 293 cells the GFP-p42IP4, a chimera of human p42IP4 and green fluorescence protein (GFP). There, we studied the influence of thrombin, which raises Ins(1,3,4,5)P4, on membrane translocation of GFP-p42IP4, induced by epidermal growth factor (EGF). Thrombin in the presence of LiCl inhibited the EGF-induced membrane recruitment of GFP-p42IP4. In the absence of LiCl, thrombin weakened the EGF-mediated membrane recruitment of GFP-p42IP4. Furthermore, the participation of p42IP4 protein domains on the EGF-mediated membrane translocation was analyzed. We used several p42IP4 variants, in which one of the domains was deleted. Alternatively, single p42IP4 domain-GFP fusion proteins were generated. Only the p42IP4 variant lacking the ZF domain showed a very weak membrane translocation in response to EGF stimulation, but all the other p42IP4 variants did not translocate. Thus, we conclude that the combination of both PH domains with ZF is required for membrane translocation of p42IP4. [ABSTRACT FROM AUTHOR] |
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