p42IP4/Centaurin α1, a Brain-specific PtdIns(3,4,5)P3/Ins(1,3,4,5)P4-binding Protein: Membrane Trafficking Induced by Epidermal Growth Factor is Inhibited by Stimulation of Phospholipase C-coupled Thrombin Receptor

Abstract

The brain-specific 42-kDa protein, p42(IP4), contains a N-terminal zinc finger (ZF) motif and a tandem of two pleckstrin homology (PH) domains. p42(IP4) 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-p42(IP4), a chimera of human p42(IP4) 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-p42(IP4), induced by epidermal growth factor (EGF). Thrombin in the presence of LiCl inhibited the EGF-induced membrane recruitment of GFP-p42(IP4). In the absence of LiCl, thrombin weakened the EGF-mediated membrane recruitment of GFP-p42(IP4). Furthermore, the participation of p42(IP4) protein domains on the EGF-mediated membrane translocation was analyzed. We used several p42(IP4) variants, in which one of the domains was deleted. Alternatively, single p42(IP4) domain-GFP fusion proteins were generated. Only the p42(IP4) variant lacking the ZF domain showed a very weak membrane translocation in response to EGF stimulation, but all the other p42(IP4) variants did not translocate. Thus, we conclude that the combination of both PH domains with ZF is required for membrane translocation of p42(IP4).

RanBPM, a novel interaction partner of the brain‐specific protein p42IP4/centaurin α‐1

“…Cell culture methods were done as already published before (Sedehizade et al 2002(Sedehizade et al , 2005. For cell transfection magnetassisted transfection was performed according to manufacturer's instruction (IBA GmbH, Göttingen, Germany).…”

“…Otherwise, the GFPtagged protein behaved like the native protein. For instance, it is translocated to plasma membrane and into the nucleus in cell stimulation experiments with epidermal growth factor and nerve growth factor (Sedehizade et al 2005). Therefore, we assumed the GFP-tagged protein to be functional and properly folded.…”

“…It is not completely understood whether both PH domains have to act together for ligand binding or whether they may bind independently of each other. Within the ARFGAP domain there is a conserved zinc finger motif which is assumed to be responsible for nucleo-/cytoplasmic shuttling (Sedehizade et al 2005). p42 IP4 can shuttle between cytosol, nucleus and plasma membrane, depending on the cell stimulation situation .…”

The phosphorylation status of extracellular‐regulated kinase 1/2 in astrocytes and neurons from rat hippocampus determines the thrombin‐induced calcium release and ROS generation

“…1996; Wang et al. 2002b) and, conversely, was shown to inhibit epidermal growth factor‐induced membrane trafficking of p42 IP4 /centaurin α1, a protein with specific binding sites for phosphatidylinositol 3,4,5‐trisphosphate and inositol 1,3,4,5‐tetrakisphosphate (Sedehizade et al. 2005; Haase et al.…”

Regulators and Effectors of Arf GTPases in Neutrophils

“…The ADAP subfamily includes two structurally related proteins with an Arf GAP and two PH domains in tandem [ 127 ]. ADAP1 is a brain specific PtdIns(3,4,5)P 3 -binding protein that functions as an Arf6 GAP in vivo [ 172 , 173 ]. ADAP1 also serves as a scaffold in several signalling pathways through interaction with proteins such as F-actin, the kinesin family protein K1F13B, Ran binding protein in microtubule organizing center, α -tubulin, and PKC family members to name a few (reviewed in [ 174 ]).…”