He Q, Dent E W, Meiri K F
Department of Pharmacology, SUNY Health Science Center, Syracuse, New York 13210, USA.
J Neurosci. 1997 May 15;17(10):3515-24. doi: 10.1523/JNEUROSCI.17-10-03515.1997.
Synthesis of GAP-43 (also known as neuromodulin) in neurons is induced during axon growth, and high concentrations (estimated between 50 and 100 microM) accumulate in the growth cone. GAP-43 is tightly associated with the growth cone membrane skeleton, the structure that transduces extracellular guidance cues into alterations in morphology by spatially regulating polymerization of actin filaments, thereby causing directional changes in axon growth. GAP-43 cosediments with actin filaments, and its phosphorylation on serine 41 by PKC, too, is spatially regulated so that phosphorylated GAP-43 is found in areas where growth cones make productive, stable contacts with other cells. In contrast, unphosphorylated GAP-43, which binds calmodulin, is always found in parts of the growth cone that are retracting. Here we have used a cell-free assay to investigate how the phosphorylation status of GAP-43 affects its interactions with actin and show that both phosphorylated and unphosphorylated GAP-43 have different, independent effects on actin filament structure. Phosphorylated GAP-43 stabilizes long actin filaments (Kd = 161 nM), and antibodies to phosphorylated GAP-43 inhibit binding of actin to phalloidin, implying a lateral interaction with filaments. In contrast, unphosphorylated GAP-43 reduces filament length distribution (Kd = 1.2 microM) and increases the critical concentration for polymerization. Prebinding calmodulin potentiates this effect. The results show that spatially regulated post-translational modifications of GAP-43 within the growth cone, which can be regulated in response to extracellular signals, have the ability to directly influence the structure of the actin cytoskeleton.
轴突生长过程中会诱导神经元合成GAP - 43（也称为神经调节蛋白），高浓度（估计在50至100微摩尔之间）的GAP - 43会在生长锥中积累。GAP - 43与生长锥膜骨架紧密相关，该结构通过在空间上调节肌动蛋白丝的聚合，将细胞外导向信号转化为形态变化，从而引起轴突生长的方向改变。GAP - 43与肌动蛋白丝一起沉降，其丝氨酸41位点被蛋白激酶C磷酸化的过程也受到空间调节，使得磷酸化的GAP - 43出现在生长锥与其他细胞形成有效、稳定接触的区域。相反，结合钙调蛋白的未磷酸化GAP - 43总是出现在生长锥回缩的部分。在这里，我们使用无细胞分析方法来研究GAP - 43的磷酸化状态如何影响其与肌动蛋白的相互作用，并表明磷酸化和未磷酸化的GAP - 43对肌动蛋白丝结构具有不同的独立影响。磷酸化的GAP - 43可稳定长肌动蛋白丝（解离常数Kd = 161纳摩尔），针对磷酸化GAP - 43的抗体可抑制肌动蛋白与鬼笔环肽的结合，这意味着它与肌动蛋白丝存在侧向相互作用。相比之下，未磷酸化的GAP - 43会减少肌动蛋白丝长度分布（Kd = 1.2微摩尔）并增加聚合的临界浓度。预先结合钙调蛋白会增强这种作用。结果表明，生长锥内GAP - 43的空间调节翻译后修饰能够响应细胞外信号进行调节，并直接影响肌动蛋白细胞骨架的结构。
