Phosphorylation-dependent binding of a synthetic MARCKS peptide to calmodulin.
Phosphorylation-dependent binding of a synthetic MARCKS peptide to calmodulin.
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DOI：
10.1016/s0021-9258(19)67742-2
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发表时间：
1991-03
期刊：
影响因子：
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通讯作者：
B. McIlroy;J. Walters;P. Blackshear;J. Johnson
中科院分区：
文献类型：
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作者：
B. McIlroy;J. Walters;P. Blackshear;J. Johnson
合成了一种 25 个氨基酸的肽，包含四个蛋白激酶 C (PKC) 磷酸化位点和肉豆蔻酰化富含丙氨酸的 C 激酶底物 (MARCKS) 蛋白的钙调蛋白 (CaM) 结合域，并用于确定磷酸化对其结合和 CaM 调节的影响。该肽的 PKC 磷酸化（3.0 mol Pi/mol 肽）导致其对 CaM 的亲和力降低 200 倍。肽的 PKC 磷酸化导致其在一段时间内与 CaM 解离，该时间过程与 1 mol 丝氨酸/mol 肽的磷酸化平行。该肽抑制 CaM 与肌球蛋白轻链激酶的结合以及 CaM 对磷酸二酯酶和钙调神经磷酸酶的刺激。肽的 PKC 磷酸化导致结合的 CaM 快速释放，使其随后与肌球蛋白轻链激酶 (t1/2 = 1.6 分钟) 结合，刺激磷酸二酯酶 (t1/2 = 1.2 分钟) 和钙调神经磷酸酶 (t1/2 = 1.7 分钟)。部分纯化的 MARCKS 蛋白对 CaM 磷酸二酯酶产生类似的抑制作用，这种抑制作用可被 PKC 磷酸化逆转。肽的 PKC 磷酸化主要发生在丝氨酸 8 和丝氨酸 12 处，丝氨酸 12 的磷酸化调节肽对 CaM 的亲和力。因此，肽和 MARCKS 蛋白的 PKC 磷酸化导致 CaM 快速释放以及随后 CaM 依赖性酶的激活。这个过程可能允许 PKC 和 CaM 依赖性信号转导途径之间的相互作用。
A 25-amino acid peptide, containing the four protein kinase C (PKC) phosphorylation sites and the calmodulin (CaM) binding domain of the myristoylated alanine-rich C kinase substrate (MARCKS) protein, has been synthesized and used to determine the effects of phosphorylation on its binding and regulation of CaM. PKC phosphorylation of this peptide (3.0 mol of Pi/mol of peptide) produced a 200-fold decrease in its affinity for CaM. PKC phosphorylation of the peptide resulted in its dissociation from CaM over a time course that paralleled the phosphorylation of 1 mol of serine/mol of peptide. The peptide inhibited CaM's binding to myosin light chain kinase and CaM's stimulation of phosphodiesterase and calcineurin. PKC phosphorylation of the peptide resulted in a rapid release of bound CaM, allowing its subsequent binding to myosin light chain kinase (t1/2 = 1.6 min), stimulation of phosphodiesterase (t1/2 = 1.2 min) and calcineurin (t1/2 = 1.7 min). Partially purified MARCKS protein produced a similar inhibition of CaM-phosphodiesterase which was reversed by PKC phosphorylation. PKC phosphorylation of the peptide occurred primarily at serine 8 and serine 12, and phosphorylation of serine 12 regulated peptide affinity for CaM. Thus, PKC phosphorylation of the peptide and the MARCKS protein results in the rapid release of CaM and the subsequent activation of CaM-dependent enzymes. This process might allow for interplay between PKC and CaM-dependent signal transduction pathways.