Anthropological and clinical implications for the structural diversity of the CagA oncoprotein. enables pathological interaction of CagA with host SH2 domain\containing proteins. Most notably, CagA forms a complex with the SHP2 tyrosine phosphatase, which contains 2 SH2 domains in its N\terminal region, through tyrosine\phosphorylated EPIYA\C or EPIYA\D segments. 6 Importantly, SHP2 binds to EPIYA\D with 2 orders of magnitude stronger affinity than to EPIYA\C. 8 The CagA\SHP2 interaction leads to deregulation of the SHP2 phosphatase activity, which is essential for full activation of the RAS\ERK signaling pathway. 4 , 9 As SHP2 is a prooncogenic phosphatase, 10 CagA has been considered to promote gastric carcinogenesis at least partly by aberrant activation of SHP2. Although PIs are minor Dichlorophene components of plasma membrane lipids, they are crucial for fundamental cellular processes, including cell signaling, membrane trafficking, and cytoskeletal rearrangements. 11 , 12 Metabolic abnormalities of PIs cause various diseases, especially cancer. For example, gene amplification or gain\of\function mutation of and loss\of\function mutation of have been shown to be associated with a diverse array of cancers. 13 , 14 Both SHIP1 and its homologue SHIP2 are phosphatidylinositol 5\phosphatases that contain a single SH2 domain in their N\terminal regions. 15 , 16 , 17 , 18 The major role Rabbit polyclonal to ADO of these lipid phosphatases is to dephosphorylate PI(3,4,5)P3 at the 5\position and convert it to PI(3,4)P2. Although the expression of SHIP1 is restricted to hematopoietic cells, SHIP2 is more ubiquitously expressed. 17 , 18 SHIP2 is diffusely distributed to the cytoplasm and is translocalized to the plasma membrane upon growth factor stimuli. 16 SHIP2 plays a critical role in local cytoskeletal rearrangement that mediates focal adhesion turnover, generation of podosomes, or lamellipodia formation in response to a growth factor by increasing the local concentration of membranous PI(3,4)P2, which interacts with several PH domain\containing proteins, including lamellipodin and TAPP1/2, and Dichlorophene thereby causes actin cytoskeletal rearrangements. 19 , 20 Independently of its catalytic function, SHIP2 also acts as a protein scaffold that binds to actin\related proteins such as p130Cas and filamin, which regulate cell adhesion and membrane ruffling. 21 , 22 These interactions also play substantial roles in cell adhesion and migration. The SH2 domain of SHIP2 has been shown to interact with ITIMs. 23 Interestingly, 1 of the SH2 domain\containing proteins that are also known to interact with ITIM motifs is SHP2, 24 suggesting that the SH2 domains of SHP2 and SHIP2 share binding targets in common. Indeed, in this study, we found that SHIP2 binds to the tyrosine\phosphorylated EPIYA\C or EPIYA\D segments of CagA through the SH2 domain. Following complex formation, CagA tethers SHIP2 to the plasma membrane and thereby increases the level of membranous PI(3,4)P2, which could strengthen the attachment of to gastric epithelial cells and thereby enhance the delivery of CagA into the host cells, which would enhance the formation of the oncogenic CagA\SHP2 complex. 2.?MATERIALS AND METHODS 2.1. Cells and transfection All cell lines have been reported previously 4 , 25 and were tested for mycoplasma contamination by PCR prior to use. Human gastric cancer\derived gastric epithelial AGS cells and nontransformed human gastric epithelial GES\1 cells were cultured in RPMI\1640 medium supplemented with 10% FBS. Monkey kidney COS\7 cells were cultured in DMEM with 10% FBS. Cells were transfected with expression vectors using Lipofectamine 2000 reagent (Invitrogen) Dichlorophene according to the manufacturers instructions. gene KO cell.