Molecular Characterization and Regulation of GTP-binding Proteins

BIG 1激活人adp核糖基化因子（ARF） 1和3，通过加速GTP取代ARF结合的GDP，启动膜泡形成的外壳蛋白募集。用BIG1抗体对HepG2细胞的共沉淀（IP）蛋白进行分析，发现了激酶家族成员21A (KIF21A)，这是一种正端定向马达蛋白，可将货物从微管组织中心移走。内源蛋白的互IP及其过表达c端区域的胞内相互作用与KIF21A- BIG1相互作用一致。干扰细胞中ARF1的循环激活和失活改变了BIG1的分布及其与KIF21A的相互作用。这些新发现的BIG1和KIF21A的相互作用使我们进一步研究了BIG1和BIG2在细胞运动和定向中的功能，这显然涉及2D和3D系统中细胞的不同事件和信号通路。最初，我们的一个主要重点是用培养细胞进行二维伤口愈合试验，这种方法可以从几个方面区分对运动至关重要的因素和那些根据内部或外部线索影响细胞极化/方向的因素。这样的研究将使我们能够更好地理解它们的机制，通过这些机制，它们可以将膜运输中的局部事件与更远距离的运输过程结合起来，并将这些过程与BIG蛋白的各种信号传导和支架功能联系起来。

BIG 1 activates human ADP-ribosylation factors (ARF) 1 and 3 by accelerating the replacement of ARF-bound GDP with GTP to initiate recruitment of coat proteins for membrane vesicle formation. Analysis of proteins that co-precipitated (IP) from HepG2 cells with BIG1 antibodies identified kinesin family member 21A (KIF21A), a plus-end-directed motor protein that moves cargo away from the microtubule-organizing center on microtubules. Reciprocal IP of endogenous proteins and intracellular interaction of their over-expressed C-terminal regions were consistent with KIF21A- BIG1 interaction. Interference with cyclic activation and inactivation of ARF1 in cells altered the distribution of BIG1 as well as its interaction with KIF21A. These newly recognized interactions of BIG1 and KIF21A led us to investigate further the functions of BIG1 and BIG2 in cell movement and orientation, which clearly involve different events and signaling pathways for cells in 2D and 3D systems. Initially, one major focus for us has been on 2D wound-healing assays with cultured cells that allow distinction in several ways among factors critical for motility and those that influence cell polarization/orientation in response to internal or external cues. Such studies should enable us to understand better the mechanisms through which, acting together, they may integrate local events in membrane trafficking with longer-range transport processes and to relate those processes to the diverse signaling and scaffold functions of the BIG protiens. Another important subject of interest is ARD1, a 64-kDa protein with an ARF domain at the C-terminus that we discovered and characterized >15 years ago. We later demonstrated its E3 ubiquitin ligase activity, but understanding the intracellular function of ARD1 continues to be limited. After showing that it is the E3 ligase activity that prevents accumulation of detectable amounts of over-expressed ARD1 in cultured cells, we prepared multiple lines of ARD1-null mouse embryo fibroblasts stably expressing constructs for inducible synthesis of ARD1 WT and mutants that lack E3 ligase activity or are constitutively bound to GTP (active) or GDP(inactive). Our significant new data on ARD1 behavior and function in cells are presented in a Ms now under review. Almost a decade ago we first reported a 100-kDa ARF-activating protein termed ARF guanine nucleotide-exchange protein (GEP) 100, which preferentially activates ARF6. Other workers described a family of ARF6-activating BRAGs (brefeldin A-resistant ARF GEF), one of which, BRAG 2a, corresponds to GEP100. Someya, Moss, and Nagaoka had more recently demonstrated a role for GEP 100 in apoptosis of mononuclear phagocytes, particularly interesting because it appeared independent of the ARF-activating Sec 7 domain. This year, they described a role for GEP100 in phagocytosis. Stable depletion of GEP100 decreased phagocytosis of serum-treated zymosan and IgG-coated latex beads by human monocyte-macrophage-like U937 cells differentiated with PMA. The impaired phagocytic activity was not restored by GEP100 delta Sec 7, a mutant lacking the ARF-activating Sec 7 domain. GEP100-depleted cells exhibited also a reduction in F-actin fibers surrounding internalized particles, although attachment of those particles to cells and amounts of C3bi and Fcy receptors were not affected. On immunofluorescence microscopy, GEP100 and ARF6 appeared concentrated and partially co-localized around internalized particles. Knock-down of ARF6 had no further effect on phagocytosis by GEP100-depleted cells. Their phagocytic activity was, however, rescued by expression of the constitutively active ARF6 Q67N mutant, but not by the dominant negative ARF6 T27N mutant. These data are consistent with the conclusion that GEP100 functions in phagocytosis via its role in ARF6-dependent actin remodeling.