[Effects of lipid rafts on signal transmembrane transduction mediated by c-Met].

OBJECTIVE: To study the effects of lipid rafts on cell signal transmembrane transduction mediated by c-Met. METHODS: After HepG2Cells were treated with MbCD to disrupt the lipid rafts and were treated with artificial recombination hepatocyte growth factor to activate c-Met, the activities of PLCr1/PKC, PI3K/Akt and MAPK signaling pathways in HepG2 cells were analyzed using Western blot. RESULTS: (1) After disruption of lipid rafts with MbCD, phosphorylation of PLCr1 decreased by 35% (P = 0.022); the content of PLCr in the cytoplasm increased by 1.75 fold (P = 0.017); PLCr1 conjugated with membrane decreased by 30% (P = 0.037). (2) The content of PKB in the cytosol decreased by 38% (P = 0.028), and the phosphorylation level of PKB conjugated with membrane decreased by 14% (P = 0.041). At the same time, PDK translocation from cytosol to the plasma membrane and its activation were inhibited by treatment with MbCD. (3) Treatment with MbCD had no significant effect on ErK/MAPK, p38/MAPK and JNK/MAPK signaling pathways. CONCLUSION: Disruption of lipid rafts with MbCD inhibits the activation of PLCr1/PKC and PI3K/PKB signaling pathways by HGF/cMet, but has no effect on MAPK signaling pathway.

Electrospray ionization mass spectrometry as a critical tool for revealing new properties of snake venom phospholipase A2.

Results from high-performance liquid chromatography/nano-electrospray ionization tandem mass spectrometry (HPLC/nESI-MS/MS) coupled to two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE) indicated that the monomer and dimer of phospholipase A(2) (PLA(2)) coexisted in crude Chinese Agkistrodon blomhoffii Ussurensis snake venom (ABUSV). Then, an acidic PLA(2) with the accurate molecular mass of 13979.6 Da was purified from ABUSV (mo-ABUSV-aPLA(2)). MS/MS-derived peptides from ABUSV-aPLA(2) were compared with other homologous snake venom PLA(2)s, which in turn showed that ABUSV-aPLA(2) is a novel snake venom PLA(2). Meanwhile, the ABUSV-aPLA(2) dimer (di-ABUSV-aPLA(2)) was also obtained. MS/MS analysis identified the same peptides from di-ABUSV-aPLA(2) as from mo-ABUSV-aPLA(2), which indicates that di-ABUSV-aPLA(2) is a homodimer. One Ca(2+) ion is contained per ABUSV-aPLA(2). The Ca(2+) ion is critical for both the hydrolytic activity and the structure of ABUSV-aPLA(2). Pro-Q Emerald and Pro-Q Diamond specific glycoprotein and phosphoprotein staining combined with MS/MS analysis indicated that the ABUSV-aPLA(2) is both a glycoprotein and a phosphoprotein, which to our knowledge is the first such report for a snake venom PLA(2) and thus provides new threads for the study of the functions and structures of snake venom PLA(2)s. One phosphorylation site and the size of the glycan chain are determined by using HPLC/nESI-MS/MS and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. The delicate utilization of ESI-MS can exert tremendous impact on protein sciences.