研究微管上之PKA在神經突型態發生中之功能

Abstract

神經細胞藉由神經突型態發生(neurite morphogenesis)而生長出軸突與樹突。在這個過程中，微管細胞骨架扮演了相當關鍵的角色。我們先前的蛋白質體研究顯示，在神經分化和神經突型態發生後，蛋白質激酶A (PKA) 在微管上的含量會顯著地提升。雖然目前已知多種細胞機制都需要PKA參與其中，但PKA在神經突型態發生中組織微管結構的功能目前仍不清楚。因為PKA參與了許多種細胞機制，直接抑制PKA蛋白質表現將會造成許多和神經突型態發生無關的表現型(phenotype)。為了避免此種情形，我們發展出一套能夠改變微管上PKA含量卻不影響胞內整體PKA表現量的方法。此方法讓我們得以檢驗PKA在微管上的功能。 MAP2c是一種神經元獨有的微管蛋白，在神經元中也是依靠MAP2c將PKA固著在微管上。已知大量表現MAP2c會使非神經元細胞產生細胞突 (cellular protrusion)。我們將MAP2c中的PKA binding domain移除掉後構築了MAP2c-∆RII-EGFP；大量表現MAP2c-∆RII-EGFP能使我們在不影響PKA表現量的情況下降低微管上的PKA含量。本研究也發現，在P19細胞中大量表現MAP2c-∆RII-EGFP會導致細胞長出較短的細胞突（相較於大量表現MAP2c的細胞）。此外，由PKA binding domain和微管蛋白（tubulin)構築出的融合蛋白PKABD-Tub能夠將內生性的PKA帶到微管上。有了該融合蛋白，我們便不需使用突變的MAP2c來將PKA帶到微管上。表現PKABD-Tub1的P19細胞相較於表現EGFP-Tub1的細胞會有較長的細胞突。以上結果顯示在非神經細胞中，位於微管上的PKA能增強細胞突的延伸能力。 此外，在小鼠初代海馬迴神經元中大量表現MAP2c-EEE-EGFP比過量表現MAP2c-EEE-∆RII-EGFP有更短的神經突。而大量表現由PKA binding domain 和增強綠色螢光蛋白(EGFP)構築出的融合蛋白PKABD-EGFP也會使初代海馬迴神經元表現較短的神經突(與單獨表現EGFP的初代海馬迴神經元之神經突相比)。這些結果顯示PKA和微管的結合也在神經突延伸 (neurite elongation)上有重要的功能。

Neurite morphogenesis is a process through which neurons generate their widespread axon and dendrites. During neurite morphogenesis, the microtubule cytoskeleton plays a crucial role. Our proteomic study indicated that the amount of protein kinase A (PKA) on microtubules significantly increased after neuronal differentiation and neurite morphogenesis. PKA has been shown to play essential roles in various cellular processes. However, its function on microtubule organization during neurite morphogenesis remains elusive. Since PKA is essential for various cellular functions, knocking down PKA genes will causes a plethora of phenotypes unrelated to neurite morphogenesis. To circumvent this problem, we developed methods to specifically alter the amount of PKA on microtubules without changing the overall PKA level. This allows us to determine the specific function of PKA on microtubule. MAP2c, a neuron-specific microtubule-associated protein, is also a PKA anchoring protein in neurons. It has been shown that overexpressing MAP2c-EGFP can produce cellular protrusion in non-neuronal cells. A MAP2c truncation construct (MAP2c-∆RII-EGFP) is generated in which the PKA binding domain was removed; this allows us to reduce PKA localization on microtubules without changing the overall PKA level. Here we show that overexpressing MAP2c-∆RII-EGFP in P19 cells resulted in shorter cell protrusions than those overexpressing MAP2c-EGFP. Furthermore, a fusion protein of PKA-binding domain and tubulin (PKABD-Tub) was constructed to recruit endogenous PKA onto microtubules. This fusion construct enabled us to alter the association between PKA and microtubules without using the MAP2c mutants. P19 cells expressing PKABD-Tub construct possessed longer cell protrusions than those expressing EGFP-Tub control. These results suggest that the recruitment of PKA on microtubules can enhance the elongation of cell protrusions in non-neuronal cells. In addition, overexpressing MAP2c-EEE-EGFP (a construct in which the microtubule-binding domain was mutated to prevent it from binding to the microtubules) in mouse primary hippocampal neurons resulted in shorter neurite length than MAP2c-EEE-∆RII-EGFP. Furthermore, a similar effect of reducing neurite length in neurons overexpressing a fusion of PKA-binding domain and EGFP (PKABD-EGFP) than EGFP was observed in mouse primary hippocampal neurons. These results indicated that PKA’s association with microtubules is important for neurite elongation as well.