Regulation of brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1) and BIG2 activity via PKA and protein phosphatase 1γ
Regulation of brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1) and BIG2 activity via PKA and protein phosphatase 1γ
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DOI：
10.1073/pnas.0611696104
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发表时间：
2007-02-27
影响因子：
11.1
通讯作者：
Vaughan, Martha
中科院分区：
文献类型：
--
作者：
Kuroda, Fuminobu;Moss, Joel;Vaughan, Martha
布雷菲德菌素A抑制的鸟嘌呤核苷酸交换蛋白（GEPs）BIG 1和BIG 2激活囊泡运输所需的ADP-核糖基化因子（ARF）GTP酶。两种分子都含有一个或多个结合蛋白激酶A的位点，即，激酶锚定蛋白（AKAP）序列。细胞cAMP的升高导致PKA催化的BIG 1磷酸化和核积累，但不是BIG 2。然后我们询问BIG 1磷酸化是否改变了其GEP活性。BIG 1或BiG 2与PKA催化亚基和ATP孵育导致其电泳迁移延迟，与PKA磷酸化一致。冈田酸抑制许多蛋白磷酸酶，包括蛋白磷酸酶1（PP 1）和PP 2A，可以逆转PKA催化的磷酸化。用冈田酸孵育HepG 2细胞引起推测磷酸化的BIG 1和BIG 2的浓度依赖性积累，并降低了迁移率，随后用特异性重组磷酸酶体外孵育增加了迁移率，PP 1 γ> PP 2A>> PP 1 α。对于GEP活性的测定，通过使用特异性siRNA从已经分别耗尽BIG 2和BIG 1的细胞中免疫沉淀BIG 1和BIG 2。每一个的GEP活性在与重组PKA加ATP孵育后显著降低，并且通过与PP 1 γ孵育恢复。与PP 1 γ在调节BIG中的作用一致，内源性PP 1 γ，而不是PP 1 α或β，与来自微粒体组分的BIG 1或BIG 2免疫沉淀。所有观察结果均与BIG 1和BIG 2磷酸化对囊泡运输的影响一致，通过改变ARF激活以及cAMP、PKA和PIP 1 γ在BIG 1和BIG 2激活ARF中的调节作用。
Brefeldin A-inhibited guanine nucleotide-exchange proteins (GEPs) BIG1 and BIG2 activate ADP-ribosylation factor (ARF) GTPases, which are required for vesicular trafficking. Both molecules contain one or more sites for binding protein kinase A, i.e., A kinase-anchoring protein (AKAP) sequences. Elevation of cell cAMP caused PKA-catalyzed phosphorylation and nuclear accumulation of BIG1 but not BIG2. We then asked whether BIG1 phosphorylation altered its GEP activity. Incubation of BIG1 or BiG2 with PKA catalytic subunits and ATP resulted in retardation of their electrophoretic migration, consistent with PKA phosphorylation. Okadaic acid inhibits many protein phosphatases, including protein phosphatase 1 (PP1) and PP2A, that can reverse PKA-catalyzed phosphorylation. Incubation of HepG2 cells with okadaic acid caused concentration-dependent accumulation of presumably phosphorylated BIG1 and BIG2 with decreased mobility, which was increased by subsequent incubation in vitro with specific recombinant phosphatases, PP1 gamma > PP2A >> PP1 alpha. For assays of GEP activity, BIG1 and BIG2 were immunoprecipitated from cells that had been depleted, respectively, of BIG2 and BIG1 by using specific siRNA. GEP activity of each was significantly decreased after incubation with recombinant PKA plus ATP and restored by incubation with PP1 gamma. In agreement with a role for PP1 gamma in regulation of BIG, endogenous PP1 gamma, but not PP1 alpha or beta, was immunoprecipitated with BIG1 or BIG2 from microsomal fractions. All observations are consistent with the effects of BIG1 and BIG2 phosphorylation on vesicular trafficking, via alterations in ARF activation and regulatory roles for cAMP, PKA, and PIP1 gamma in ARF activation by BIG1 and BIG2.