p190RhoGAP and Rap-dependent RhoGAP (ARAP3) inactivate RhoA in response to nerve growth factor leading to neurite outgrowth from PC12 cells

Rat pheochromocytoma (PC12) cells have been used to investigate neurite outgrowth. Nerve growth factor (NGF) has been well known to induce neurite outgrowth from PC12 cells. RhoA belongs to Ras-related small GTP-binding proteins, which regulate a variety of cellular processes, including cell morphology alteration, actin dynamics, and cell migration. NGF suppressed GTP-RhoA levels after 12 h in PC12 cells and was consistently required for a long time to induce neurite outgrowth. Constitutively active (CA)-RhoA suppressed neurite outgrowth from PC12 cells in response to NGF, whereas dominant-negative (DN)-RhoA stimulated it, suggesting that RhoA inactivation is essential for neurite outgrowth. Here, we investigated the mechanism of RhoA inactivation. DN-p190RhoGAP abrogated neurite outgrowth, whereas wild-type (WT)-p190RhoGAP and WT-Src synergistically stimulated it along with accelerating RhoA inactivation, suggesting that p190RhoGAP, which can be activated by Src, is a major component in inhibiting RhoA in response to NGF in PC12 cells. Contrary to RhoA, Rap1 was activated by NGF, and DN-Rap1 suppressed neurite outgrowth, suggesting that Rap1 is also essential for neurite outgrowth. RhoA was co-immunoprecipitated with Rap1, suggesting that Rap1 interacts with RhoA. Furthermore, a DN-Rap-dependent RhoGAP (ARAP3) prevented RhoA inactivation, abolishing neurite formation from PC12 cells in response to NGF. These results suggest that NGF activates Rap1, which, in turn, up-regulates ARAP3 leading to RhoA inactivation and neurite outgrowth from PC12 cells. Taken together, p190RhoGAP and ARAP3 seem to be two main factors inhibiting RhoA activity during neurite outgrowth in PC12 cells in response to NGF.

Effects of adenovirus vector expression hFRNK on phosphorylated 190RhoGAP and RhoA Activity in colo320WT colorectal cells / 中国实用内科杂志

Objective To study effects of hFRNK gene on phosphorylated p190RhoGAP expression and RhoA activation by mediated adenoviral vector in colorectal carcinoma cell Colo320WT stimulated with extrinsic gastrin17 in vitro.Methods AdEasyTMsystem was used to construct pAdhFRNK expressing human FRNK gene by recombination in E.coli.BJ5283.pCR3.1/GR plasmid expressing gastrin receptor CCK-2 was transfected into colonic carcinoma cell line Colo320 cells by LipofectamineTM2000 and expression stably CCK-2R clones was selected by G418(500 ?g/mL).The expression levels of gastrin receptor of Colo320 cells and the transfected cells Colo320WT were assayed by RT-PCR.Colo320WT cells were treated by 10～8 mol/L Gastrin17 for 0h and 12 h;and after Colo320WT cells were infected by pAdhFRNK(MOI:100)for 2 d,the cells were treated by Gastrin17 for 12 h again.The expression levels of phosphorylated p190RhoGAP of Colo320WT cells were assayed by immuniprecipation and western blot.RhoA activation was assayed by pull-down.using GST-Rhotekin-RBD followed by RhoA blotting.Results When 10～8 mol/L Gastrin17 stimulated Colo320WT cells for 12 h,the expression levels of phosphorylated p190RhoGAP increased apparently and RhoA activation diminished.When pAdhFRNK infected Colo320WT cells for 2 d and 10～8 mol/L Gastrin17 treated the cells for 12 h,the expression levels of phosphorylated p190RhoGAP decreased apparently and RhoA activity was elevated.Conclusion hFRNK can inhibit expression of phosphorylated p190RhoGAP and enhanced RhoA activity in the cells stimulated with Gastrin17,and its mechanism is probably that hFRNK can block FAK phosphorylation and FAK pathway.