A single residue in the C1 domain sensitizes novel protein kinase C isoforms to cellular diacylglycerol production
A single residue in the C1 domain sensitizes novel protein kinase C isoforms to cellular diacylglycerol production
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DOI：
10.1074/jbc.c600268200
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发表时间：
2007-01-12
影响因子：
4.8
通讯作者：
Newton, Alexandra C.
中科院分区：
文献类型：
--
作者：
Dries, Daniel R.;Gallegos, Lisa L.;Newton, Alexandra C.
C1结构域介导常规和新型蛋白激酶C（PKC）亚型的二酰基甘油（DAG）依赖性易位。在新型PKC亚型（nPKC）中，该结构域以足够高的亲和力结合膜，以在不存在任何其他靶向机制的情况下将nPKC募集到膜上。然而，在常规PKC（cPKC）亚型中，C1结构域对DAG的亲和力低两个数量级，需要C1结构域和Ca2+调节的C2结构域的协调结合以进行易位和激活。在这里，我们确定了一个单一的残基，调谐DAG（而不是佛波酯）含有膜的C1b域的亲和力。该残基在cPKC的C1b结构域中与Tyr一样不变，在所有其他PKC C1结构域中与Trp一样不变。结合使用模型膜的研究，以及活细胞成像研究的黄色荧光蛋白标记的C1域，揭示了色氨酸与酪氨酸之间切换的C1域的物种具有足够高的亲和力，以响应激动剂产生的DAG的一个是不能响应生理水平的DAG。此外，我们表明，虽然酪氨酸在这个开关位置的C1结构域在未刺激的条件下引起胞质定位，色氨酸的目标，这些领域的高尔基体，可能是由于在这个地区的DAG的基础水平。因此，在C1结构域的这个关键位置上，Trp与Tyr控制着PKC的膜亲和力和定位。单个残基控制C1结构域对含DAG的膜的亲和力的发现为以下原因提供了分子解释：1）单独的DAG足以激活nPKC而不是cPKC，以及2）nPKC靶向高尔基体。
The C1 domain mediates the diacylglycerol (DAG)-dependent translocation of conventional and novel protein kinase C (PKC) isoforms. In novel PKC isoforms(nPKCs), this domain binds membranes with sufficiently high affinity to recruit nPKCs to membranes in the absence of any other targeting mechanism. In conventional PKC (cPKC) isoforms, however, the affinity of the C1 domain for DAG is two orders of magnitude lower, necessitating the coordinated binding of the C1 domain and a Ca2+-regulated C2 domain for translocation and activation. Here we identify a single residue that tunes the affinity of the C1b domain for DAG-(but not phorbol ester-) containing membranes. This residue is invariant as Tyr in the C1b domain of cPKCs and invariant as Trp in all other PKC C1 domains. Binding studies using model membranes, as well as live cell imaging studies of yellow fluorescent protein-tagged C1 domains, reveal that Trp versus Tyr toggles the C1 domain between a species with sufficiently high affinity to respond to agonist-produced DAG to one that is unable to respond to physiological levels of DAG. In addition, we show that while Tyr at this switch position causes cytosolic localization of the C1 domain under unstimulated conditions, Trp targets these domains to the Golgi, likely due to basal levels of DAG at this region. Thus, Trp versus Tyr at this key position in the C1 domain controls both the membrane affinity and localization of PKC. The finding that a single residue controls the affinity of the C1 domain for DAG- containing membranes provides a molecular explanation for why 1) DAG alone is sufficient to activate nPKCs but not cPKCs and 2) nPKCs target to the Golgi.