Molecular Characterization and Regulation of GTP-binding Proteins

外体样囊泡中cAMP对TNFRI释放的刺激依赖于两个BIG2 AKAP序列和A-激酶RII B亚单位。AKAP复合体中可能存在至少一种cAMP磷酸二酯酶(PDE)。用BIG1或BIG2抗体检测HeLa胞浆和蛋白中的PDE活性(总、PDE3和PDE4)。每个免疫沉淀物(IP)含有约4%的胞浆PDE4和15%~19%的PDE3活性，基本上都是PDE3A。共聚焦免疫荧光显微镜显示PDE3A IP含有BIG1和BIG2。 BREFELDIN A抑制的鸟嘌呤核苷酸交换蛋白(BIG)1通过加速ARF结合的GDP被GTP取代来激活人ADP核糖化因子(ARF)1和3，从而启动膜囊泡形成所需的外壳蛋白的募集。对从HepG2细胞与BIG1抗体共沉淀的蛋白质进行的液相色谱MS/MS分析确定了Kinesin家族成员21A(KIF21A)，这是一种正端定向的马达蛋白，可以将微管上的货物移离微管组织中心。内源性蛋白质的相互作用(IP)和免疫活性BIG1与核周过表达的GFP-KIF21A在显微镜下的明显重叠符合KIF21ABIG1的相互作用。KIF21A和BIG1的全长及其片段在HEK293细胞中的过表达和相互作用的IP表明，KIF21A的C-末端有7个WD-40重复序列，可能与BIG1的C-末端区域的结构相互作用。通过过表达结构性活性ARF1(Q71L)或显性失活ARF1(T31N)来干扰ARF1的周期激活和失活，改变了BIG1的分布及其与KIF21A的相互作用。对ARF1的需求通过其与siRNA的选择性耗尽而得到确认。与诺可达唑破坏微管不同，通过用特定的siRNA去除KIF21A选择性地抑制转运改变了BIG1的分布，而不改变固有高尔基膜蛋白的分布。这些新发现的BIG1和KIF21A的相互作用应该使我们能够更好地理解它们共同作用的机制，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BIG1的不同信号和支架功能联系起来。 用小干扰RNA特异性耗尽HeLa细胞PDE3A显著减少膜相关的BIG1和BIG2，在共聚焦免疫荧光显微镜下，它们从初始的核周高尔基体浓度广泛分散。同时，激活的ARF1-GTP显著减少。通过与西洛胺孵育细胞1-h选择性抑制PDE3A，类似地减少膜相关的BIG1。我们认为，降低PDE3A允许cAMP在其酶活性限制cAMP浓度的微区中积累。CAMP激活的PKA使BIG1和BIG2(用于组装PKA、PDE3A和其他分子的AKAP)磷酸化，从而降低它们的GEP活性，从而降低激活的ARF1-GTP的数量。因此，这些BIG1和BIG2 AKAP复合体中的PDE3A可能通过限制具有时间和空间特异性的cAMP效应来调节ARF的功能。

Some cAMP stimulation of TNFRI release in exosome-like vesicles depended on two BIG2 AKAP sequences as well as the A-kinase RII B subunit. The presence of at least one cAMP phosphodiesterase (PDE) in the AKAP complex was likely. PDE activities (total, PDE3, and PDE4) were assayed in samples of HeLa cytosol and proteins precipitated from it by BIG1 or BIG2 antibodies. Each immunoprecipitate (IP) contained ca 4% of cytosolic PDE4 and 15-19% of PDE3 activity, essentially all of which was identified as PDE3A. PDE3A IPs contained both BIG1 and BIG2 which also appeared partially co-localized on confocal immunofluorescence microscopy. Brefeldin A-inhibited guanine nucleotide-exchange protein (BIG) 1 activates human ADP-ribosylation factor (ARF) 1 and 3 by accelerating the replacement of ARF-bound GDP with GTP to initiate recruitment of coat proteins for membrane vesicle formation. Liquid chromatography MS/MS analysis of proteins that co-precipitated from HepG2 cells with BIG1 antibodies identified kinesin family member 21A (KIF21A), a plus-end-directed motor protein that moves cargo on microtubules away from the microtubule-organizing center. Reciprocal (IP) of endogenous proteins and microscopically apparent overlap of immunoreactive BIG1 with overexpressed GFP-KIF21A in the perinuclear region were consistent with KIF21ABIG1 interaction. Overexpression of full-length KIF21A and BIG1 and their fragments in HEK293 cells followed by reciprocal IP revealed that the C-terminal tail of KIF21A, with seven WD-40 repeats, may interact with structure in the C-terminal region of BIG1. Interfering with cyclic activation and inactivation of ARF1 by overexpressing constitutively active ARF1(Q71L) or dominant inactive ARF1(T31N) altered the distribution of BIG1 as well as its interaction with KIF21A. A requirement for ARF1 was confirmed by its selective depletion with siRNA. Unlike disruption of microtubules with nocodazole, selective inhibition of transport by depletion of KIF21A with specific siRNA altered BIG1 distribution without changing that of intrinsic Golgi membrane proteins. These newly recognized interactions of BIG1 and KIF21A 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 BIG1. Specific depletion of HeLa cell PDE3A with small interfering RNA significantly decreased membrane-associated BIG1 and BIG2, which by confocal immunofluorescence microscopy were widely dispersed from an initial perinuclear Golgi concentration. Concurrently, activated ARF1-GTP was significantly decreased. Selective inhibition of PDE3A by 1-h incubation of cells with cilostamide similarly decreased membrane-associated BIG1. We suggest that decreasing PDE3A allowed cAMP to accumulate in microdomains where its enzymatic activity limited cAMP concentration. cAMP-activated PKA phosphorylated BIG1 and BIG2 (AKAPs for assembly of PKA, PDE3A, and other molecules), which decreased their GEP activity and thereby amounts of activated ARF1-GTP. Thus, PDE3A in these BIG1 and BIG2 AKAP complexes may contribute to the regulation of ARF function via limitation of cAMP effects with spatial and temporal specificity.