Abstract 5803: Interaction of Docking Protein Gab1 With Tyrosine Phosphatase SHP2 Regulates Hepatocyte Growth Factor-dependent ERK5 Activation and Upregulation of KLF2 in the Vascular Endothelium

Abstract

Background : Docking protein Gab1 has been reported to have crucial roles for activation of both mitogen-acitvated protein kinase and AKT downstream of various receptor tyrosine kinases. In the last scientific session, we reported that endothelium-specific Gab1 knockout mice (Gab1ECKO) displayed severe limb necrosis after operatively-induced hindlimb ischemia (HLI) due to the defect of angiogenic response. We also reported that Gab1 was essential for activation of both extracellular signal-regulated kinase 1/2 (ERK1/2) via tyrosine phosphatase SHP2 and AKT via PI3-kinase p85 subunit in human umbilical vein endothelial cells (HUVECs) in response to hepatocyte growth factor (HGF), respectively. Here, we investigated the downstream target of the HGF/Met/Gab1-dependent signaling pathway responsible for the maintenance of endothelium. Methods and Results : We examined the effect of adenovirus-mediated overexpression of β -galactosidase ( β -gal), wild-type Gab1 (Gab1 WT ), or mutated Gab1 which can’t bind with SHP2 (Gab1 ΔSHP2 ) or p85 (Gab1 Δp85 ) on the activation of downstream signaling pathways in HUVECs. HGF-induced activation of both ERK1/2 and ERK5 was enhanced in the HUVECs expressing Gab1 WT or Gab1 Δp85 , but abrogated in those expressing Gab1 ΔSHP2 , compared with control cells expressing β -gal. HGF-induced AKT activation was specifically attenuated in cells expressing Gab1 Δp85 , compared with other three groups. Using DNA microarray analysis, we found that the stimulation with HGF upregulated the expression of Kruppel-like factor 2 (KLF2), a crucial transcriptional factor for endothelial maintenance, and thrombomodulin, a well-known target gene of KLF2, through interaction of Gab1 with SHP2 in HUVECs. Next, we examined the involvement of MEK5-ERK5 pathway for the KLF2 gene expression. HGF-dependent upregulation of KLF2 was almost abrogated by the adenoviral overexpression of either dominant-negative MEK5 or dominant-negative ERK5 in HUVECs. Furthermore, the expression of KLF2 and TM in the endothelium was significantly decreased in the Gab1ECKO, compared with control after HLI. Conclusion : Interaction of Gab1 with SHP2 is essential for both activation of ERK5 and upregulation of KLF2 in response to HGF in the endothelium.