Trivedi Niraj, Marsh Phil, Goold Robert G, Wood-Kaczmar Alison, Gordon-Weeks Phillip R
The MRC Centre for Developmental Neurobiology, New Hunts House, Guy's Campus, King's College London, London SE1 1UL, UK.
J Cell Sci. 2005 Mar 1;118(Pt 5):993-1005. doi: 10.1242/jcs.01697.
Recent experiments show that the microtubule-associated protein (MAP) 1B is a major phosphorylation substrate for the serine/threonine kinase glycogen synthase kinase-3beta (GSK-3beta) in differentiating neurons. GSK-3beta phosphorylation of MAP1B appears to act as a molecular switch regulating the control that MAP1B exerts on microtubule dynamics in growing axons and growth cones. Maintaining a population of dynamically unstable microtubules in growth cones is important for axon growth and growth cone pathfinding. We have mapped two GSK-3beta phosphorylation sites on mouse MAP1B to Ser1260 and Thr1265 using site-directed point mutagenesis of recombinant MAP1B proteins, in vitro kinase assays and phospho-specific antibodies. We raised phospho-specific polyclonal antibodies to these two sites and used them to show that MAP1B is phosphorylated by GSK-3beta at Ser1260 and Thr1265 in vivo. We also showed that in the developing nervous system of rat embryos, the expression of GSK-3beta phosphorylated MAP1B is spatially restricted to growing axons, in a gradient that is highest distally, despite the expression of MAP1B and GSK-3beta throughout the entire neuron. This suggests that there is a mechanism that spatially regulates the GSK-3beta phosphorylation of MAP1B in differentiating neurons. Heterologous cell transfection experiments with full-length MAP1B, in which either phosphorylation site was separately mutated to a valine or, in a double mutant, in which both sites were mutated, showed that these GSK-3beta phosphorylation sites contribute to the regulation of microtubule dynamics by MAP1B.
最近的实验表明，微管相关蛋白（MAP）1B是分化神经元中丝氨酸/苏氨酸激酶糖原合酶激酶-3β（GSK-3β）的主要磷酸化底物。MAP1B的GSK-3β磷酸化似乎起着分子开关的作用，调节MAP1B对生长轴突和生长锥中微管动力学的控制。在生长锥中维持一群动态不稳定的微管对于轴突生长和生长锥路径寻找很重要。我们通过对重组MAP1B蛋白进行定点诱变、体外激酶测定和磷酸特异性抗体，将小鼠MAP1B上的两个GSK-3β磷酸化位点定位到Ser1260和Thr1265。我们制备了针对这两个位点的磷酸特异性多克隆抗体，并利用它们证明MAP1B在体内被GSK-3β在Ser1260和Thr1265处磷酸化。我们还表明，在大鼠胚胎发育中的神经系统中，GSK-3β磷酸化的MAP1B的表达在空间上局限于生长轴突，呈远端最高的梯度分布，尽管MAP1B和GSK-3β在整个神经元中都有表达。这表明在分化神经元中存在一种在空间上调节MAP1B的GSK-3β磷酸化的机制。用全长MAP1B进行的异源细胞转染实验表明，其中任何一个磷酸化位点分别突变为缬氨酸，或者在双突变体中两个位点都发生突变，这些GSK-3β磷酸化位点有助于MAP1B对微管动力学的调节。
