Keating H H, Asai D J
Department of Biology, Purdue University, West Lafayette, Indiana 47907.
Dev Biol. 1994 Mar;162(1):143-53. doi: 10.1006/dbio.1994.1074.
The functional significance of microtubule-associated protein 1B (MAP1B) phosphorylation during neuronal differentiation is unknown. In the present study we examined the hypothesis that the phosphorylation of MAP1B is required for neurite outgrowth. We reasoned that if MAP1B phosphorylation was important for neurite outgrowth then the intracellular distribution of phosphorylated MAP1B might exist as a discrete subset of the pattern for total MAP1B. We utilized a monoclonal antibody (mAb 7-1.1) that specifically recognizes a phosphorylated epitope on MAP1B and a polyclonal antiserum that recognizes all MAP1B protein to compare the distributions of phosphorylated and total MAP1B during neurite outgrowth. Phosphorylated MAP1B progressively accumulated in both the soluble and cytoskeletal fractions of differentiating cells. Similar proportions of total and phosphorylated MAP1B were associated with the cytoskeletons of differentiating PC12 cells. Within individual cells, phosphorylated MAP1B, in comparison with total MAP1B, was not limited to a particular intracellular domain. Phosphorylated MAP1B was present in both neurites and cell bodies. It was associated with fibrillar microtubules in neurites and growth cones, but it appeared nonfibrillar within cell bodies. In some cells that differentiated rapidly, there was little phosphorylated MAP1B in the early neurites despite the presence of extensive microtubules. In addition, although phosphorylated MAP1B increased in populations of mature PC12 cell cultures, increases in phosphorylated MAP1B did not always correlate with neurite outgrowth in individual cells. These results suggest that the phosphorylated isoform of MAP1B recognized by mAb 7-1.1 may not be required for neurite outgrowth.
微管相关蛋白1B（MAP1B）磷酸化在神经元分化过程中的功能意义尚不清楚。在本研究中，我们检验了一个假设，即MAP1B的磷酸化是神经突生长所必需的。我们推断，如果MAP1B磷酸化对神经突生长很重要，那么磷酸化MAP1B的细胞内分布可能作为总MAP1B模式的一个离散子集存在。我们使用了一种特异性识别MAP1B上磷酸化表位的单克隆抗体（mAb 7-1.1）和一种识别所有MAP1B蛋白的多克隆抗血清，以比较神经突生长过程中磷酸化和总MAP1B的分布。磷酸化MAP1B在分化细胞的可溶性和细胞骨架部分中逐渐积累。总MAP1B和磷酸化MAP1B与分化的PC12细胞的细胞骨架相关的比例相似。在单个细胞内，与总MAP1B相比，磷酸化MAP1B并不局限于特定的细胞内区域。磷酸化MAP1B存在于神经突和细胞体中。它与神经突和生长锥中的纤维状微管相关，但在细胞体内似乎是非纤维状的。在一些快速分化的细胞中，尽管存在大量微管，但早期神经突中磷酸化MAP1B很少。此外，虽然成熟PC12细胞培养群体中磷酸化MAP1B增加，但磷酸化MAP1B的增加并不总是与单个细胞中的神经突生长相关。这些结果表明，mAb 7-1.1识别的MAP1B磷酸化异构体可能不是神经突生长所必需的。
