Glycogen synthase kinase-3beta phosphorylation of MAP1B at Ser1260 and Thr1265 is spatially restricted to growing axons.
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.
Trivedi
Niraj
N
The MRC Centre for Developmental Neurobiology, New Hunts House, Guy's Campus, King's College London, London SE1 1UL, UK.
Marsh
Phil
P
Goold
Robert G
RG
Wood-Kaczmar
Alison
A
Gordon-Weeks
Phillip R
PR
D000818:Animals; D001369:Axons / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D049109:Cell Proliferation; D002540:Cerebral Cortex / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004622:Embryo, Mammalian / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D018507:Gene Expression Regulation, Developmental; D005982:Glutathione Transferase / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D015151:Immunoblotting; D051379:Mice; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009474:Neurons / Q000378:metabolism; D009841:Oligonucleotides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry*; D013912:Threonine / Q000737:chemistry*; D013997:Time Factors; D014162:Transfection; D014633:Valine / Q000737:chemistry
D018076:DNA, Complementary; D000939:Epitopes; D008869:Microtubule-Associated Proteins; D009841:Oligonucleotides; D010455:Peptides; D011994:Recombinant Proteins; C085284:microtubule-associated protein 1B; D013912:Threonine; D012694:Serine; D005982:Glutathione Transferase; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; C000605508:Gsk3b protein, rat; D038362:Glycogen Synthase Kinase 3; D014633:Valine