Goold R G, Owen R, Gordon-Weeks P R
Developmental Biology Research Centre, The Randall Institute, King's College London, London WC2B 5RL, UK.
J Cell Sci. 1999 Oct;112 ( Pt 19):3373-84. doi: 10.1242/jcs.112.19.3373.
We have recently shown that glycogen synthase kinase 3beta (GSK3beta) phosphorylates the microtubule-associated protein (MAP) 1B in an in vitro kinase assay and in cultured cerebellar granule cells. Mapping studies identified a region of MAP1B high in serine-proline motifs that is phosphorylated by GSK3beta. Here we show that COS cells, transiently transfected with both MAP1B and GSK3beta, express high levels of the phosphorylated isoform of MAP1B (MAP1B-P) generated by GSK3beta. To investigate effects of MAP1B-P on microtubule dynamics, double transfected cells were labelled with antibodies to tyrosinated and detyrosinated tubulin markers for stable and unstable microtubules. This showed that high levels of MAP1B-P expression are associated with the loss of a population of detyrosinated microtubules in these cells. Transfection with MAP1B protected microtubules in COS cells against nocodazole depolymerisation, confirming previous studies. However, this protective effect was greatly reduced in cells containing high levels of MAP1B-P following transfection with both MAP1B and GSK3beta. Since we also found that MAP1B binds to tyrosinated, but not to detyrosinated, microtubules in transfected cells, we propose that MAP1B-P prevents tubulin detyrosination and subsequent conversion of unstable to stable microtubules and that this involves binding of MAP1B-P to unstable microtubules. The highest levels of MAP1B-P are found in neuronal growth cones and therefore our findings suggest that a primary role of MAP1B-P in growing axons may be to maintain growth cone microtubules in a dynamically unstable state, a known requirement of growth cone microtubules during pathfinding. To test this prediction, we reduced the levels of MAP1B-P in neuronal growth cones of dorsal root ganglion cells in culture by inhibiting GSK3beta with lithium. In confirmation of the proposed role of MAP1B-P in maintaining microtubule dynamics we found that lithium treatment dramatically increased the numbers of stable (detyrosinated) microtubules in the growth cones of these neurons.
我们最近发现，在体外激酶分析以及培养的小脑颗粒细胞中，糖原合酶激酶3β（GSK3β）可使微管相关蛋白（MAP）1B发生磷酸化。图谱研究确定了MAP1B中一个富含丝氨酸 - 脯氨酸基序的区域，该区域可被GSK3β磷酸化。在此我们表明，同时瞬时转染了MAP1B和GSK3β的COS细胞，表达由GSK3β产生的高水平磷酸化形式的MAP1B（MAP1B - P）。为了研究MAP1B - P对微管动力学的影响，用针对酪氨酸化和去酪氨酸化微管蛋白标记物的抗体对双重转染的细胞进行标记，以区分稳定和不稳定微管。结果表明，高水平的MAP1B - P表达与这些细胞中一部分去酪氨酸化微管的丢失有关。转染MAP1B可保护COS细胞中的微管免受诺考达唑解聚作用的影响，这证实了先前的研究。然而，在同时转染了MAP1B和GSK3β后含有高水平MAP1B - P的细胞中，这种保护作用大大降低。由于我们还发现MAP1B在转染细胞中与酪氨酸化微管结合，但不与去酪氨酸化微管结合，我们提出MAP1B - P可阻止微管蛋白去酪氨酸化以及随后不稳定微管向稳定微管的转变，并且这涉及MAP1B - P与不稳定微管的结合。在神经元生长锥中发现了最高水平的MAP1B - P，因此我们的研究结果表明，MAP1B - P在生长轴突中的主要作用可能是使生长锥微管维持在动态不稳定状态，这是生长锥微管在路径寻找过程中的一个已知需求。为了验证这一预测，我们通过用锂抑制GSK3β来降低培养的背根神经节细胞神经元生长锥中MAP1B - P的水平。为了证实MAP1B - P在维持微管动力学中的作用，我们发现锂处理显著增加了这些神经元生长锥中稳定（去酪氨酸化）微管的数量。
