Ziemba Brian P, Burke John E, Masson Glenn, Williams Roger L, Falke Joseph J
Molecular Biophysics Program, University of Colorado, Boulder, Colorado; Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado.
Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.
Biophys J. 2016 Apr 26;110(8):1811-1825. doi: 10.1016/j.bpj.2016.03.001.
In chemotaxing ameboid cells, a complex leading-edge signaling circuit forms on the cytoplasmic leaflet of the plasma membrane and directs both actin and membrane remodeling to propel the leading edge up an attractant gradient. This leading-edge circuit includes a putative amplification module in which Ca(2+)-protein kinase C (Ca(2+)-PKC) is hypothesized to phosphorylate myristoylated alanine-rich C kinase substrate (MARCKS) and release phosphatidylinositol-4,5-bisphosphate (PIP2), thereby stimulating production of the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3) by the lipid kinase phosphoinositide-3-kinase (PI3K). We investigated this hypothesized Ca(2+)-PKC-MARCKS-PIP2-PI3K-PIP3 amplification module and tested its key predictions using single-molecule fluorescence to measure the surface densities and activities of its protein components. Our findings demonstrate that together Ca(2+)-PKC and the PIP2-binding peptide of MARCKS modulate the level of free PIP2, which serves as both a docking target and substrate lipid for PI3K. In the off state of the amplification module, the MARCKS peptide sequesters PIP2 and thereby inhibits PI3K binding to the membrane. In the on state, Ca(2+)-PKC phosphorylation of the MARCKS peptide reverses the PIP2 sequestration, thereby releasing multiple PIP2 molecules that recruit multiple active PI3K molecules to the membrane surface. These findings 1) show that the Ca(2+)-PKC-MARCKS-PIP2-PI3K-PIP3 system functions as an activation module in vitro, 2) reveal the molecular mechanism of activation, 3) are consistent with available in vivo data, and 4) yield additional predictions that are testable in live cells. More broadly, the Ca(2+)-PKC-stimulated release of free PIP2 may well regulate the membrane association of other PIP2-binding proteins, and the findings illustrate the power of single-molecule analysis to elucidate key dynamic and mechanistic features of multiprotein signaling pathways on membrane surfaces.
在进行趋化运动的阿米巴样细胞中，一个复杂的前沿信号传导回路在质膜的细胞质小叶上形成，并指导肌动蛋白和膜重塑，以推动前沿沿着吸引剂梯度向上移动。这个前沿回路包括一个假定的放大模块，其中钙蛋白激酶C（Ca(2+)-PKC）被假定磷酸化富含肉豆蔻酰化丙氨酸的C激酶底物（MARCKS）并释放磷脂酰肌醇-4,5-二磷酸（PIP2），从而刺激脂质激酶磷酸肌醇-3-激酶（PI3K）产生信号脂质磷脂酰肌醇-3,4,5-三磷酸（PIP3）。我们研究了这个假定的Ca(2+)-PKC-MARCKS-PIP2-PI3K-PIP3放大模块，并使用单分子荧光测量其蛋白质成分的表面密度和活性来测试其关键预测。我们的研究结果表明，Ca(2+)-PKC和MARCKS的PIP2结合肽共同调节游离PIP2的水平，PIP2既是PI3K的对接靶点又是底物脂质。在放大模块的关闭状态下，MARCKS肽隔离PIP2，从而抑制PI3K与膜的结合。在开启状态下，MARCKS肽的Ca(2+)-PKC磷酸化逆转PIP2隔离，从而释放多个PIP2分子，这些分子将多个活性PI3K分子招募到膜表面。这些发现1）表明Ca(2+)-PKC-MARCKS-PIP2-PI3K-PIP3系统在体外作为一个激活模块发挥作用，2）揭示了激活的分子机制，3）与现有的体内数据一致，4）产生了可在活细胞中测试的额外预测。更广泛地说，Ca(2+)-PKC刺激的游离PIP2释放很可能调节其他PIP2结合蛋白的膜结合，这些发现说明了单分子分析在阐明膜表面多蛋白信号通路的关键动态和机制特征方面的强大作用。
