Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093, Warsaw, Poland.
Neurochem Res. 2013 Mar;38(3):519-29. doi: 10.1007/s11064-012-0944-5. Epub 2012 Dec 9.
Palmitoylcarnitine was observed previously to promote differentiation of neuroblastoma NB-2a cells, and to affect protein kinase C (PKC). Palmitoylcarnitine was also observed to increase palmitoylation of several proteins, including a PKC substrate, whose expression augments during differentiation of neural cells-a growth associated protein GAP-43, known to bind phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)]. Since palmitoylated proteins are preferentially localized in sphingolipid- and cholesterol-rich microdomains of plasma membrane, the present study has been focused on a possible effect of palmitoylcarnitine on GAP-43 localization in these microdomains. Palmitoylcarnitine treatment resulted in GAP-43 appearance in floating fractions (rafts) in sucrose gradient and increased co-localization with cholesterol and with PI(4,5)P(2), although co-localization of both lipids decreased. GAP-43 disappeared from raft fraction upon treatment with 2-bromopalmitate (an inhibitor of palmitoylating enzymes) and after treatment with etomoxir (carnitine palmitoyltransferase I inhibitor). Raft localization of GAP-43 was completely abolished by treatment with methyl-β-cyclodextrin, a cholesterol binding agent, while there was no change upon sequestration of PI(4,5)P(2) with neomycin. GAP-43 co-precipitated with a monomeric form of Gα(o), a phenomenon diminished after palmitoylcarnitine treatment and paralleled by a decrease of Gα(o) in the raft fraction. These observations point to palmitoylation of GAP-43 as a mechanism leading to an increased localization of this protein in microdomains of plasma membrane rich in cholesterol, in majority different, however, from microdomains in which PI(4,5)P(2) is present. This localization correlates with decreased interaction with Gα(o) and suppression of its activity-an important step regulating neural cell differentiation.
先前的研究表明，软脂酰肉碱能促进神经母细胞瘤 NB-2a 细胞的分化，并影响蛋白激酶 C（PKC）。软脂酰肉碱还观察到增加几种蛋白质的棕榈酰化，包括 PKC 底物，其表达在神经细胞分化过程中增加 - 一种与生长相关的蛋白 GAP-43，已知与磷脂酰肌醇 4,5-二磷酸 [PI（4,5）P（2）] 结合。由于棕榈酰化蛋白优先定位于质膜的鞘脂和胆固醇丰富的微区，因此本研究集中于软脂酰肉碱对 GAP-43 在这些微区中的定位的可能影响。软脂酰肉碱处理导致 GAP-43 出现在蔗糖梯度的漂浮部分（筏）中，并且与胆固醇和 PI（4,5）P（2）的共定位增加，尽管这两种脂质的共定位减少。用 2-溴软脂酸（棕榈酰化酶抑制剂）处理后，GAP-43 从筏部分消失，并用 etomoxir（肉碱棕榈酰转移酶 I 抑制剂）处理后消失。用甲基-β-环糊精处理可完全消除 GAP-43 的筏定位，而用新霉素隔离 PI（4,5）P（2）时则没有变化。GAP-43 与单体形式的 Gα（o）共沉淀，这一现象在用软脂酰肉碱处理后减弱，并且与 Gα（o）在筏部分的减少相平行。这些观察结果表明 GAP-43 的棕榈酰化是一种导致该蛋白在富含胆固醇的质膜微区中定位增加的机制，然而，这种定位与存在 PI（4,5）P（2）的微区不同。这种定位与与 Gα（o）的相互作用减少和其活性抑制相关 - 这是调节神经细胞分化的重要步骤。
