GEP100/Arf6 Is Required for Epidermal Growth Factor-Induced ERK/Rac1 Signaling and Cell Migration in Human Hepatoma HepG2 Cells

BACKGROUND: Epidermal growth factor (EGF) signaling is implicated in the invasion and metastasis of hepatoma cells. However, the signaling pathways for EGF-induced motility of hepatoma cells remain undefined. METHODOLOGY/PRINCIPAL FINDINGS: We found that EGF dose-dependently stimulated the migration of human hepatoma cells HepG2, with the maximal effect at 10 ng/mL. Additionally, EGF increased Arf6 activity, and ectopic expression of Arf6 T27N, a dominant negative Arf6 mutant, largely abolish EGF-induced cell migration. Blocking GEP100 with GEP100 siRNA or GEP100-△PH, a pleckstrin homology (PH) domain deletion mutant of GEP100, blocked EGF-induced Arf6 activity and cell migration. EGF also increased ERK and Rac1 activity. Ectopic expression GEP100 siRNA, GEP100-△PH, or Arf6-T27N suppressed EGF-induced ERK and Rac1 activity. Furthermore, blocking ERK signaling with its inhibitor U0126 remarkably inhibited both EGF-induced Rac1 activation as well as cell migration, and ectopic expression of inactive mutant form of Rac1 (Rac1-T17N) also largely abolished EGF-induced cell migration. CONCLUSIONS/SIGNIFICANCE: Taken together, this study highlights the function of the PH domain of GEP100 and its regulated Arf6/ERK/Rac1 signaling cascade in EGF-induced hepatoma cell migration. These findings could provide a rationale for designing new therapy based on inhibition of hepatoma metastasis

Purification of the 260 kDa cytosolic complex involved in the Superoxide production of guinea pig neutrophils

AbstractA 260 kDa cytosolic complex (SP-1) was purified from guinea pig neutrophils. SP-1 was composed of 63 kDa, 47 kDa and 39 kDa proteins. The 63 kDa and 47 kDa proteins proved to correspond to human p67phox and p47phox by Western blot analysis, whereas Western blot and amino acid sequence analyses revealed that the 39 kDa protein was a novel protein. The 47 kDa protein was separated from the 63 kDa and 39 kDa proteins by dithiothreitol (DTT)-treatment. On the other hand, the 63 kDa and 39 kDa proteins were not separated with DTT, detergent and ethanol treatment. These results suggest that the 39 kDa protein tightly associates with the 63 kDa protein and may regulate the function of the 63 kDa protein

Characterization of cDNA clones encoding guinea pig neutrophil cationic peptides

AbstractcDNA clones encoding antimicrobial guinea pig neutrophil cationic peptides GNCP-1 and GNCP-2 were isolated from a bone marrow cell cDNA library. Analysis of these clones indicated that both GNCPs were produced as precursor proteins comprising 93 amino acid residues, which were composed of signal sequences (N-terminal 19 residues), pro-peptide sequences (43 residues) and mature GNCP sequences (31 residues). The deduced amino acid sequence showed that there were only two amino acid differences between GNCP-1 and GNCP-2, one in the pro-peptide region and one in the mature peptide region. Interestingly, Northern blot analysis and transcription run-off assay revealed that the expression of GNCP mRNA and the transcription of GNCP gene was observed in bone marrow cells but not in mature neutrophils. These observations suggest that mature neutropils, despite their abundant content of GNCPs, lose the capacity to synthesize GNCPs

Translocation of guinea pig p40-phox during activation of NADPH oxidase

AbstractThe superoxide-producing NADPH oxidase consists of membrane-associated cytochrome b558 and cytosolic components, p47-phox and p67-phox. Recently, we have found a novel cytosolic component, p40-phox, which is tightly associated with p67-phox. In this study, we examined the translocation of p40-phox during activation of NADPH oxidase in a cell-free system using the membrane and the purified p47-phox/p67-phox/p40-phox complex. p40-phox was translocated to the membrane by arachidonic acid in a dose-dependent manner. The translocation pattern of p40-phox was similar to those of p47-phox and p67-phox. However, immunoprecipitation assay revealed that p40-phox was dissociated from p47-phox and p67-phox during activation. The translocation of three cytosolic components was not affected by the deletion of GTP-γ-s from the reaction mixture. Interestingly, a synthetic peptide corresponding to carboxyl-terminus of p40-phox inhibited the activation of NADPH oxidase and translocation of p40-phox, p47-phox and p67-phox, suggesting that p40-phox might play a role in the activation of NADPH oxidase. These observations suggest that p40-phox is dissociated from p67-phox during activation, and translocates to the membrane by GTP-γ-s-independent mechanism