Corbin John A, Evans John H, Landgraf Kyle E, Falke Joseph J
Molecular Biophysics Program, and Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
Biochemistry. 2007 Apr 10;46(14):4322-36. doi: 10.1021/bi062140c. Epub 2007 Mar 17.
The C2 domain is a ubiquitous, conserved protein signaling motif widely found in eukaryotic signaling proteins. Although considerable functional diversity exists, most C2 domains are activated by Ca2+ binding and then dock to a specific cellular membrane. The C2 domains of protein kinase Calpha (PKCalpha) and cytosolic phospholipase A2alpha (cPLA2alpha), for example, are known to dock to different membrane surfaces during an intracellular Ca2+ signal. Ca2+ activation targets the PKCalpha C2 domain to the plasma membrane and the cPLA2alpha C2 domain to the internal membranes, with no detectable spatial overlap. It is crucial to determine how such targeting specificity is achieved at physiological bulk Ca2+ concentrations that during a typical signaling event rarely exceed 1 muM. For the isolated PKCalpha C2 domain in the presence of physiological Ca2+ levels, the target lipids phosphatidylserine (PS) and phosphatidylinositol-4,5-bisphosphate (PIP2) are together sufficient to recruit the PKCalpha C2 domain to a lipid mixture mimicking the plasma membrane inner leaflet. For the cPLA2alpha C2 domain, the target lipid phosphatidylcholine (PC) appears to be sufficient to drive membrane targeting to an internal membrane mimic at physiological Ca2+ levels, although the results do not rule out a second, unknown target molecule. Stopped-flow kinetic studies provide additional information about the fundamental molecular events that occur during Ca2+-activated membrane docking. In principle, C2 domain-directed intracellular targeting, which requires coincidence detection of multiple signals (Ca2+ and one or more target lipids), can exhibit two different mechanisms: messenger-activated target affinity (MATA) and target-activated messenger affinity (TAMA). The C2 domains studied here both utilize the TAMA mechanism, in which the C2 domain Ca2+ affinity is too low to be activated by physiological Ca2+ signals in most regions of the cell. Only when the C2 domain nears its target membrane, which provides a high local concentration of target lipid, is the effective Ca2+ affinity increased by the coupled binding equilibrium to a level that enables substantial Ca2+ activation and target docking. Overall, the findings emphasize the importance of using physiological ligand concentrations in targeting studies because super-physiological concentrations can drive docking interactions even when an important targeting molecule is missing.
C2结构域是一种普遍存在且保守的蛋白质信号基序，广泛存在于真核生物信号蛋白中。尽管存在相当大的功能多样性，但大多数C2结构域通过结合Ca2+而被激活，然后与特定的细胞膜对接。例如，蛋白激酶Cα（PKCα）和胞质磷脂酶A2α（cPLA2α）的C2结构域在细胞内Ca2+信号传导过程中会与不同的膜表面对接。Ca2+激活将PKCα C2结构域靶向质膜，将cPLA2α C2结构域靶向内膜，没有可检测到的空间重叠。确定在生理状态下典型信号事件中很少超过1μM的Ca2+浓度下，如何实现这种靶向特异性至关重要。对于在生理Ca2+水平存在下分离的PKCα C2结构域，靶脂质磷脂酰丝氨酸（PS）和磷脂酰肌醇-4,5-二磷酸（PIP2）共同足以将PKCα C2结构域募集到模拟质膜内小叶的脂质混合物中。对于cPLA2α C2结构域，靶脂质磷脂酰胆碱（PC）似乎足以在生理Ca2+水平下驱动膜靶向至内膜模拟物，尽管结果并未排除第二种未知的靶分子。停流动力学研究提供了有关Ca2+激活的膜对接过程中发生的基本分子事件的更多信息。原则上，需要同时检测多种信号（Ca2+和一种或多种靶脂质）的C2结构域导向的细胞内靶向可表现出两种不同的机制：信使激活的靶亲和力（MATA）和靶激活的信使亲和力（TAMA）。本文研究的C2结构域均利用TAMA机制，其中C2结构域对Ca2+的亲和力过低，在细胞的大多数区域无法被生理Ca2+信号激活。只有当C2结构域接近其靶膜时，靶膜提供高局部浓度的靶脂质，通过与靶脂质的偶联结合平衡，有效Ca2+亲和力才会增加到能够实现大量Ca2+激活和靶对接的水平。总体而言，这些发现强调了在靶向研究中使用生理配体浓度的重要性，因为超生理浓度即使在缺少重要靶向分子时也能驱动对接相互作用。
