The closure of Pak1-dependent macropinosomes requires the phosphorylation of CtBP1/BARS

Abstract

Membrane fission is an essential process in membrane trafficking and other cellular functions. While many fissioning and trafficking steps are mediated by the large GTPase dynamin, some fission events are dynamin independent and involve C-terminal-binding protein-1/ brefeldinA-ADP ribosylated substrate (CtBP1/BARS). To gain an insight into the molecular mechanisms of CtBP1/ BARS in fission, we have studied the role of this protein in macropinocytosis, a dynamin-independent endocytic pathway that can be synchronously activated by growth factors. Here, we show that upon activation of the epidermal growth factor receptor, CtBP1/BARS is (a) translocated to the macropinocytic cup and its surrounding membrane, (b) required for the fission of the macropinocytic cup and (c) phosphorylated on a specific serine that is a substrate for p21-activated kinase, with this phosphorylation being essential for the fission of the macropinocytic cup. Importantly, we also show that CtBP1/BARS is required for macropinocytic internalization and infection of echovirus 1. These results provide an insight into the molecular mechanisms of CtBP1/BARS activation in membrane fissioning, and extend the relevance of CtBP1/BARSinduced fission to human viral infection.

Sequential breakdown of 3-phosphorylated phosphoinositides is essential for the completion of macropinocytosis

“…PAK-1, an effector that links RhoGTPases, such as Rac1 and Cdc42, to actin dynamics, regulates membrane ruffle formation during macropinocytosis (35)(36)(37). In the present study we found that a series of phosphoinositide phosphatases, which metabolize 3-phosphorylated phosphoinositide into PI, is required for macropinocytosis after the membrane ruffle formation.…”

“…S2B). Knockdown of either MTMR6 or MTMR9, like knockdown of PAK-1 (p21-activated kinase-1), an essential factor for macropinocytosis (35)(36)(37), significantly inhibited the dextran uptake ( Fig. 2 A and B).…”

A Raft-derived, Pak1-regulated Entry Participates in α2β1 Integrin-dependent Sorting to Caveosomes

“…Namely, in our previous study, we found that the stabilizing drug jasplakinolide had no effect for EV1 infection in CV-1 cells in contrast to the opposite results from SAOS-␣2␤1 cells here. Interestingly, our recent results showed that in SAOS-␣2␤1 cells EV1 and integrin entry are regulated by the carboxy-terminal binding protein 1/brefeldin A-ribosylated substrate (CtBP1/BARS; Liberali et al, 2008), which has been shown to regulate dynamin-independent entry in some cell lines (Bonazzi et al, 2005).…”

“…Ruthenium red staining was done as described previously (Liberali et al, 2008). Briefly, after the internalization with integrin, cells were washed in cacodylate buffer, pH 7.3, fixed, and stained with 1.3% glutaraldehyde containing 0.07 mg/ml ruthenium red in cacodylate buffer for 1 h at room temperature.…”

Unravelling the Mechanism of TrkA-Induced Cell Death by Macropinocytosis in Medulloblastoma Daoy Cells

“…To define TrkA-dependent pathways that drive macropinocytosis, we focused on molecules that serve roles in clathrin-independent endocytosis and macropinocytosis-dependent actin remodeling. Collectively, these include PAK1, PI3 kinase, and Src (32), as well as several GTPases, including H-Ras, Cdc42, Rac1, Rab5, Arf1, Arf6, RhoA, and CtBP1/BARS (15,16,21,33,34,35). With respect to Trk signaling, TrkA has been shown to activate H-Ras, Rap-1, Rac, and Cdc42 as well as to negatively regulate RhoA, depending on the cellular context (36)(37)(38)(39)(40)(41)(42)(43)(44)(45).…”