Regulation Of GTP-binding Proteins

adp核糖基化因子（ARFs）是gtp结合蛋白，可调节多种囊泡运输途径。ARF功能需要在gtp结合的活性形式和gdp结合的非活性形式之间进行调节。GTP结合由鸟嘌呤核苷酸交换蛋白（GEPs）催化，其中一些被brefeldin A （BFA，一种抑制蛋白质分泌并导致高尔基池可逆分解的药物）抑制，其中一些具有BFA抗性。该小组纯化并克隆了两个bfa抑制的GEPs （BIG1和BIG2）。内源性BIG1和BIG2通常从具有特异性抗体的培养细胞中一起沉淀，但在酵母双杂交实验中，BIG1与FKBP13相互作用，BIG2与环amp活化蛋白激酶（PKA）的调节亚基riα相互作用，它们的行为完全不同。在BIG2分子中鉴定出三个具有A激酶锚定蛋白（AKAPs）特征的序列。正在进行的研究表明，与cAMP类似物孵育的细胞核中，BIG1、BIG2和ARF都会积累，而PKA抑制可以阻止这些作用。这些发现与BIG2在协调cAMP和ARF调控通路中的作用一致。

ADP-ribosylation factors (ARFs) are GTP-binding proteins that regulate numerous vesicular trafficking pathways. ARF function requires the regulated alternation between GTP-bound active and GDP-bound inactive forms. GTP binding is catalyzed by guanine nucleotide-exchange proteins (GEPs), some of which are inhibited by brefeldin A (BFA, a drug that inhibits protein sectretion and causes reversible disintegration of Golgi cisternae) and some of which are BFA-resistant. Two BFA-inhibited GEPs (BIG1 and BIG2) had been purified and cloned by the group. Endogeneous BIG1 and BIG2 often precipitated together from cultured cells with specific antibodies, but they behaved quite differently in yeast two-hybrid experiments, where BIG1 interacted with FKBP13 and BIG2 with RIalpha, a regulatory subunit of cyclic AMP-activated protein kinase (PKA). Three sequences characteristic of A kinase-anchoring proteins (AKAPs), were identified in the BIG2 molecule. Work in progress revealed that both BIG1 and BIG2, and ARF accumulate in nuclei of cells incubated with a cAMP analogue, and these effects were prevented by PKA inhibition. These findings are consistent with a role for BIG2 in coordinating cAMP and ARF regulatory pathways. FKBP13 is an immunophilin of the FK506-binding protein family that does not bind or inhibit calcineurin. Incubation of Jurkat cells with FK506, a widely used immunosuppresant drug, increased binding of BIG1, BIG2, and ARF to Golgi and other membranes. Endogenous BIG1 in HepG2 cells accumulated in nuclei of cells incubated for 18h without serum and was further increased (and largely absent from Golgi) after 30 min exposure to FK506, which also caused movement of FKBP13 into nuclei, but not colocalized with BIG1. In serum-starved cells, BIG1 was identified in nucleoli, nuclear matrix, and pore structures. Alterations in BIG1 location, and probably functions, appear to be associated with cell cycle events, as well as exogenous signals. Characterization of RNAs associated with BIG1 in nucleoli is in progress.