Glycogen synthase kinase 3 beta phosphorylation of microtubule-associated protein 1B regulates the stability of microtubules in growth cones

We have recently shown that glycogen synthase kinase 3 beta (GSK3 beta) pho sphorylates the microtubule-associated protein (MAP) 1B in an in vitro kina se assay and in cultured cerebellar granule cells, Mapping studies identifi ed a region of MAP1B high in serine-proline motifs that is phosphorylated b y GSK3 beta. Here we show that COS cells, transiently transfected with both MAP1B and GSK3 beta, express high levels of the phosphorylated isoform of MAP1B (MAP1B-P) generated by GSK3 beta. To investigate effects of MAP1B-P o n microtubule dynamics, double transfected cells were labelled with antibod ies to tyrosinated and detyrosinated tubulin markers for stable and unstabl e microtubules. This showed that high levels of MAP1B-P expression are asso ciated with the loss of a population df detyrosinated microtubules in these cells; Transfection with MAP1B protected microtubules in COS cells against nocodazole depolymerisation, confirming previous studies, However, this pr otective effect was greatly reduced in cells containing high levels of MAP1 B-P following transfection with both MAP1B and GSK3 beta, Since we also fou nd that MAP1B binds to tyrosinated, but not to detyrosinated, microtubules in transfected cells, we propose that MAP1B-P prevents tubulin detyrosinati on and subsequent conversion of unstable to stable microtubuIes and that th is involves binding of MAP1B-P to unstable microtubules, The highest levels of MAP1B-P are found in neuronal growth cones and therefore our findings s uggest that a primary role of MAP1B-P in growing axons may be to maintain g rowth cone microtubules in a dynamically unstable state, a known requiremen t of growth cone microtubules during pathfinding, To test this prediction, we reduced the levels of MAP1B-P in neuronal growth cones of dorsal root ga nglion cells in culture by inhibiting GSK3 beta with lithium, In confirmati on of the proposed role of MAP1B-P in maintaining microtubule dynamics we f ound that lithium treatment dramatically increased the numbers of stable (d etyrosinated) microtubules in the growth cones of these neurons.