Adducin is an in vivo substrate for protein kinase C: phosphorylation in the MARCKS-related domain inhibits activity in promoting spectrin-actin complexes and occurs in many cells, including dendritic spines of neurons.
Adducin is an in vivo substrate for protein kinase C: phosphorylation in the MARCKS-related domain inhibits activity in promoting spectrin-actin complexes and occurs in many cells, including dendritic spines of neurons.
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DOI：
10.1083/jcb.142.2.485
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发表时间：
1998-07-27
期刊：
影响因子：
--
通讯作者：
Bennett V
中科院分区：
文献类型：
--
作者：
Matsuoka Y;Li X;Bennett V
内收蛋白是一种异质蛋白，其亚基含有cooh末端肉豆蔻酰基化富丙氨酸C激酶底物（MARCKS）相关结构域，该结构域覆盖并优先将谱蛋白招募到肌动蛋白细丝的快速末端。基本的marks相关结构域存在于α、β和γ内收蛋白亚基中，与钙调蛋白结合，并包含蛋白激酶C （PKC）的主要磷酸化位点。该报告首次证明了marks相关结构域的磷酸化改变了包括肌动蛋白和谱蛋白在内的内收蛋白的体外和体内活性，并且我们证明了内收蛋白是PKC或其他多种细胞类型（包括神经元）中PMA激活的激酶的重要体内底物。PKC磷酸化的天然和重组内聚蛋白抑制了肌动蛋白的封顶，并消除了内聚蛋白在肌动蛋白丝的末端和侧面招募谱蛋白的活性。一种针对rtps -丝氨酸磷酸化状态的多克隆抗体被用于评估细胞内收蛋白的磷酸化，rtps -丝氨酸是marks相关结构域中PKC的主要磷酸化位点。PMA治疗后，免疫印迹对phosphoadducin抗体的反应性在大鼠海马切片中增加了两倍，在人胚胎肾（HEK 293）细胞中增加了8 - 9倍，在MDCK细胞中增加了3倍，在人红细胞中增加了10倍以上。因此，内收蛋白的rtps -丝氨酸是PKC或其他pma活化激酶的体内磷酸化位点，而不是多种细胞类型中camp依赖性蛋白激酶的磷酸化位点。通过用野生型或PKC不磷酸化的S716A/S726A突变体α内合蛋白稳定转染MDCK细胞，研究了在marks相关区域的两个PKC磷酸化位点的生理后果。突变体α内收蛋白不再集中在细胞与细胞接触部位的细胞膜上，而是以细胞质点状分布。此外，表达α内收蛋白突变体的细胞表现出细胞质谱蛋白水平的增加，其与α内收蛋白突变体以点状模式共定位。磷酸化内收蛋白特异性抗体的免疫荧光显示在发育中的大鼠海马突触后区域内收蛋白的rtps -丝氨酸磷酸化。在培养的海马神经元树突棘中检测到高水平的磷酸腺苷。Spectrin也是树突棘的一个组成部分，尽管在不同的位置与含有磷酸腺苷的位置不同。这些数据表明，内收蛋白是多种细胞中PKC或其他pma活化激酶的重要体内底物，并且内收蛋白的磷酸化发生在树突棘中，这些树突棘被认为通过改变细胞骨架结构的形态和重组来响应外部信号。
Adducin is a heteromeric protein with subunits containing a COOH-terminal myristoylated alanine-rich C kinase substrate (MARCKS)-related domain that caps and preferentially recruits spectrin to the fast-growing ends of actin filaments. The basic MARCKS-related domain, present in α, β, and γ adducin subunits, binds calmodulin and contains the major phosphorylation site for protein kinase C (PKC). This report presents the first evidence that phosphorylation of the MARCKS-related domain modifies in vitro and in vivo activities of adducin involving actin and spectrin, and we demonstrate that adducin is a prominent in vivo substrate for PKC or other phorbol 12-myristate 13-acetate (PMA)-activated kinases in multiple cell types, including neurons. PKC phosphorylation of native and recombinant adducin inhibited actin capping measured using pyrene-actin polymerization and abolished activity of adducin in recruiting spectrin to ends and sides of actin filaments. A polyclonal antibody specific to the phosphorylated state of the RTPS-serine, which is the major PKC phosphorylation site in the MARCKS-related domain, was used to evaluate phosphorylation of adducin in cells. Reactivity with phosphoadducin antibody in immunoblots increased twofold in rat hippocampal slices, eight- to ninefold in human embryonal kidney (HEK 293) cells, threefold in MDCK cells, and greater than 10-fold in human erythrocytes after treatments with PMA, but not with forskolin. Thus, the RTPS-serine of adducin is an in vivo phosphorylation site for PKC or other PMA-activated kinases but not for cAMP-dependent protein kinase in a variety of cell types. Physiological consequences of the two PKC phosphorylation sites in the MARCKS-related domain were investigated by stably transfecting MDCK cells with either wild-type or PKC-unphosphorylatable S716A/S726A mutant α adducin. The mutant α adducin was no longer concentrated at the cell membrane at sites of cell–cell contact, and instead it was distributed as a cytoplasmic punctate pattern. Moreover, the cells expressing the mutant α adducin exhibited increased levels of cytoplasmic spectrin, which was colocalized with the mutant α adducin in a punctate pattern. Immunofluorescence with the phosphoadducin-specific antibody revealed the RTPS-serine phosphorylation of adducin in postsynaptic areas in the developing rat hippocampus. High levels of the phosphoadducin were detected in the dendritic spines of cultured hippocampal neurons. Spectrin also was a component of dendritic spines, although at distinct sites from the ones containing phosphoadducin. These data demonstrate that adducin is a significant in vivo substrate for PKC or other PMA-activated kinases in a variety of cells, and that phosphorylation of adducin occurs in dendritic spines that are believed to respond to external signals by changes in morphology and reorganization of cytoskeletal structures.