Differential functions of phospholipid binding and palmitoylation of tumour suppressor EWI2/PGRL

Abstract

The tumour suppressor EWI2 associates with tetraspanins and regulates tumour cell movement and proliferation. The short cytoplasmic domain of EWI2 is positively charged; five out of the ten residues of this domain are basic. In the present study we demonstrated that the EWI2 cytoplasmic tail interacts specifically with negatively charged PIPs (phosphatidylinositol phosphates), but not with other membrane lipids. The PIPs that interact with EWI2 cytoplasmic tail include PtdIns5P, PtdIns4P, PtdIns3P, PtdIns(3,5)P2 and PtdIns(3,4)P2. The binding affinity of PIPs to the EWI2 tail, however, is not solely based on charge because PtdIns5P, PtdIns4P and PtdIns3P have a higher affinity to EWI2 than PtdIns(3,5)P2 and PtdIns(3,4)P2 do. Mutation of either of two basic residue clusters in the EWI2 cytoplasmic tail abolishes PIP binding, and PIP binding is also determined by the position of basic residues in the EWI2 cytoplasmic tail. In addition, EWI2 is constitutively palmitoylated at the cytoplasmic cysteine residues located at the N-terminal of those basic residues. The PIP interaction is not required for, but appears to regulate, the palmitoylation, whereas palmitoylation is neither required for nor regulates the PIP interaction. Functionally, the PIP interaction regulates the stability of EWI2 proteins, whereas palmitoylation is needed for tetraspanin–EWI2 association and EWI2-dependent inhibition of cell migration and lamellipodia formation. For cell–cell adhesion and cell proliferation, the PIP interaction functions in opposition to the palmitoylation. In conclusion, the EWI2 cytoplasmic tail actively engages with the cell membrane via PIP binding and palmitoylation, which play differential roles in EWI2 functions.

Tracking Brain Palmitoylation Change: Predominance of Glial Change in a Mouse Model of Huntington’s Disease

“…The three rightmost columns provide the published evidence supporting palmitoylation documentation for each protein, including either a specific reference (see below), or a “+” indication that the protein was identified as likely palmitoylated in prior ABE- or click-based proteomic analyses (see Table S2 for references to the relevant proteomic studies and for the expanded ABE/SILAM dataset). Proteins that are expressed predominantly in either oligodendrocytes or astrocytes are underlined. References: (1) (Charych et al, 2010); (2) (Agrawal et al, 1990); (3) (Huang et al, 2010); (4) (Linder et al, 1993); (5) (He et al, 2011); (6) (Topinka and Bredt, 1998); (7) (Hayashi et al, 2005). …”

Tetraspanin-enriched microdomains regulate digitation junctions

“…Because PI-4 kinase resides in TEMs 5 , we examined PI-4 kinase product PI4P and found that PI4P molecules were localized at DJs in A431 epidermoid cancer cells and colocalized with EWI2/PGRL (Figure 6A), which as a TEM constituent physically interacts with PI4P 25 . The presence of PI4P in basal DJs was observed when the cells were removed during washes in the staining process.…”

“…Cells used in this study include human microvascular endothelial cells (HMECs) (CDC, Atlanta, GA), Du145 and LnCap human prostate cancer (ATCC, Manassas, VA), U87 human glioblastoma (ATCC), and A431 human epidermoid cancer cell lines (ATCC), Du145-Mock and -CD82 stable transfectants 18,48 , HT1080-Mock and HT1080-CD9 stable transfectants, PC3 human prostate cancer cells (ATCC) in which EWI2/PGRL was transiently silenced with siRNA oligo against the target sequence GUUCUCCUAUGCUGUCUU of EWI2/PGRL or in which control siRNA oligo was transfected 22 , MDCK-GFP:CD82 and -GFP:CD151 stable transfectants expressing the fusion proteins in which eGFP moieties were fused to the N-termini of CD82 and CD151 proteins, respectively, and NIH3T3-EWI2/PGRL transfectant 25 . The EWI2/PGRL or control siRNAs were transfected into PC3 cells with Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA) in Opti-MEM medium (Invitrogen), and the cells were used for experiments at 48 or 72 hours after the transfections.…”

IGSF3 mutation identified in patient with severe COPD alters cell function and motility

“…Cells deficient in IGSF3 had markedly increased attachment to fibronectin and decreased ability to migrate to close a scratch wound, whereas cells with increased levels of IGSF3 had increased wound repair rates. The effect on cell adhesion and mobility may be related to the ability of IGSF3 to modulate cytoskeletal function through EWI and is therefore shared with other IGSF family members such as EWI-2, CD101, and PTGFRN (26,27). In addition, the increased adhesion could be the result of changes in receptor density due to abnormal integrin anchorage or lateral mobility induced by the marked changes in sphingolipids (28) in IGSF3-deficient cells.…”