Calcium-sensitive interaction between calmodulin and modified forms of rat brain neurogranin/RC3
Calcium-sensitive interaction between calmodulin and modified forms of rat brain neurogranin/RC3
复制标题
DOI：
10.1021/bi000336l
复制
发表时间：
2000-06-20
期刊：
影响因子：
2.9
通讯作者：
McPhie, P
中科院分区：
文献类型：
--
作者：
Huang, KP;Huang, FL;McPhie, P
神经颗粒蛋白（NC）在其IQ结构域与钙调蛋白（CaM）的结合对Ca 2+浓度以及蛋白激酶C（PKC）和氧化剂的修饰敏感。NG的PKC磷酸化位点在IQ结构域内，而四个氧化剂敏感的Cys残基在该区域外。这些Cys残基被氧化形成两对分子内二硫键，并且还可以被S-亚硝基谷胱甘肽化，导致每个NG掺入四个谷胱甘肽。圆二色性（CD）表明，修饰NG的磷酸化，氧化形成分子内二硫化物，或谷胱甘肽不影响α-螺旋含量的这种蛋白质。四个Cys残基[Cys（-）-NG]突变为Gly和Ser也不影响α-螺旋含量。在无钙溶液中，CaM与还原（红）-、谷胱甘肽（GS）-或Cys（-）-NG的相互作用导致通过CD光谱测定的cx螺旋度增加，但与氧化NG（ox-NG）或磷酸化NG（PO 4-NG）的变化相对较小。用CD光谱法和沉降平衡法测定了不同修饰形式的NG与CaM的结合亲和力：Cys（-）-NG > red-NG，CS-NG > ox-NC > PO_4-NG。与Cys（-）-、red-和GS-NG不同，ox-和PO 4-NG均不与CaM亲和柱结合。因此，氧化NG形成分子内二硫化物和PKC磷酸化NG都能有效调节细胞内CaM水平。这些结果表明，修饰NG以在IQ结构域外形成分子内二硫键提供了调节其与CaM结合亲和力的替代机制。
Neurogranin (NC) binding of calmodulin (CaM) at its IQ domain is sensitive to Ca2+ concentration and to modifications by protein kinase C (PKC) and oxidants. The PKC phosphorylation site of NG is within the IQ domain whereas the four oxidant-sensitive Cys residues are outside this region. These Cys residues were oxidized forming two pairs of intramolecular disulfides, and could also be glutathiolated by S-nitrosoglutathione resulting in the incorporation of four glutathiones per NG. Circular dichroism (CD) showed that modification of NG by phosphorylation, oxidation forming intramolecular disulfides, or glutathiolation did not affect the alpha-helical content of this protein. Mutation of the four Cys residues [Cys(-)-NG] to Gly and Ser did not affect the alpha-helical content either. Interaction of CaM with the reduced (red)-, glutathiolated (GS)-, or Cys(-)-NG in the Ca2+-free solution resulted in an increase in the cx-helicity determined by their CD spectra, but relatively little change was seen with the oxidized NG (ox-NG) or phosphorylated NG (PO4-NG). The binding affinities between the various modified forms of NG and CaM were determined by CD spectrometry and sedimentation equilibrium: their affinities were Cys(-)-NG > red-NG, CS-NG > ox-NC > PO4-NG. Unlike Cys(-)-, red-, and GS-NG, neither ox- nor PO4-NG bound to a CaM-affinity column. Thus, both oxidation of NG to form intramolecular disulfides and phosphorylation of NG by PKC are effective in modulating the intracellular level of CaM. These results indicate that modification of NG to form intramolecular disulfides outside the IQ domain provides an alternative mechanism fur regulation of its binding affinity to CaM.