Johnstone M, Goold R G, Fischer I, Gordon-Weeks P R
Developmental Biology Research Centre, Randall Institute, Kings College, London, UK.
J Anat. 1997 Aug;191 ( Pt 2)(Pt 2):229-44. doi: 10.1046/j.1469-7580.1997.19120229.x.
Microtubules are important for the growth and maintenance of stable neuronal processes and their organisation is controlled partly by microtubule-associated proteins (MAPs). MAP 1B is the first MAP to be expressed in neurons and plays an important role in neurite outgrowth. MAP 1B is phosphorylated at multiple sites and it is believed that the function of the protein is regulated by its phosphorylation state. We have shown that the monoclonal antibody (mAb) RT97, which recognises phosphorylated epitopes on neurofilament proteins, fetal tau, and on Alzheimer's paired helical filament-tau, also recognises a developmentally regulated phosphorylation epitope on MAP 1B. In the rat cerebellum, Western blot analysis shows that mAb RT97 recognises the upper band of the MAP 1B doublet and that the amount of this epitope peaks very early postnatally and decreases with increasing age so that it is absent in the adult, despite the continued expression of MAP 1B in the adult. We confirmed that mAb RT97 binds to MAP 1B by showing that it recognises MAP 1B immunoprecipitated from postnatal rat cerebellum using polyclonal antibodies to recombinant MAP 1B proteins. We established that the RT97 epitope on MAP 1B is phosphorylated by showing that antibody binding was abolished by alkaline phosphatase treatment of immunoblots. Epitope mapping experiments suggest that the mAb RT97 site on MAP 1B is near the N-terminus of the molecule. Despite our immunoblotting data, immunostaining of sections of postnatal rat cerebellum with mAb RT97 shows a staining pattern typical of neurofilaments with no apparent staining of MAP 1B. For instance, basket cell axons and axons in the granule cell layer and white matter stained, whereas parallel fibres did not. These results suggest that the MAP 1B epitope is masked or lost under the immunocytochemical conditions in which the cerebellar sections are prepared. The upper band of the MAP 1B doublet is believed to be predominantly phosphorylated by proline-directed protein kinases (PDPKs). PDPKs are also good candidates for phosphorylating neurofilament proteins and tau and therefore we postulate that the sites recognised by RT97 on these neuronal cytoskeletal proteins may be phosphorylated by similar kinases. Important goals are to determine the precise location of the RT97 epitope on MAP 1B and the kinase responsible.
微管对于稳定的神经元突起的生长和维持很重要，其组织部分受微管相关蛋白（MAPs）控制。MAP 1B是第一个在神经元中表达的MAP，在神经突生长中起重要作用。MAP 1B在多个位点被磷酸化，人们认为该蛋白的功能受其磷酸化状态调节。我们已经表明，识别神经丝蛋白、胎儿tau蛋白以及阿尔茨海默病双螺旋丝tau蛋白上磷酸化表位的单克隆抗体（mAb）RT97，也识别MAP 1B上一个受发育调节的磷酸化表位。在大鼠小脑中，蛋白质印迹分析表明，mAb RT97识别MAP 1B双峰的上带，并且该表位的量在出生后极早期达到峰值，随后随着年龄增长而减少，以至于在成年大鼠中不存在，尽管成年大鼠中MAP 1B仍持续表达。我们通过显示mAb RT97识别使用针对重组MAP 1B蛋白的多克隆抗体从出生后大鼠小脑中免疫沉淀的MAP 1B，证实了mAb RT97与MAP 1B结合。我们通过显示免疫印迹经碱性磷酸酶处理后抗体结合被消除，确定了MAP 1B上的RT97表位被磷酸化。表位定位实验表明，MAP 1B上的mAb RT97位点靠近该分子的N端。尽管有我们的免疫印迹数据，但用mAb RT97对出生后大鼠小脑切片进行免疫染色显示出典型的神经丝染色模式，而MAP 1B没有明显染色。例如，篮状细胞轴突以及颗粒细胞层和白质中的轴突被染色，而平行纤维未被染色。这些结果表明，在制备小脑切片的免疫细胞化学条件下，MAP 1B表位被掩盖或丢失。MAP 1B双峰的上带被认为主要由脯氨酸导向蛋白激酶（PDPKs）磷酸化。PDPKs也是使神经丝蛋白和tau蛋白磷酸化的良好候选激酶，因此我们推测RT97在这些神经元细胞骨架蛋白上识别的位点可能由相似的激酶磷酸化。重要的目标是确定MAP 1B上RT97表位的确切位置以及负责的激酶。
