Nonprimed and DYRK1A-primed GSK3 beta-phosphorylation sites on MAP1B regulate microtubule dynamics in growing axons.
MAP1B is a developmentally regulated microtubule-associated phosphoprotein that regulates microtubule dynamics in growing axons and growth cones. We used mass spectrometry to map 28 phosphorylation sites on MAP1B, and selected for further study a putative primed GSK3 beta site and compared it with two nonprimed GSK3 beta sites that we had previously characterised. We raised a panel of phosphospecific antibodies to these sites on MAP1B and used it to assess the distribution of phosphorylated MAP1B in the developing nervous system. This showed that the nonprimed sites are restricted to growing axons, whereas the primed sites are also expressed in the neuronal cell body. To identify kinases phosphorylating MAP1B, we added kinase inhibitors to cultured embryonic cortical neurons and monitored MAP1B phosphorylation with our panel of phosphospecific antibodies. These experiments identified dual-specificity tyrosine-phosphorylation-regulated kinase (DYRK1A) as the kinase that primes sites of GSK3 beta phosphorylation in MAP1B, and we confirmed this by knocking down DYRK1A in cultured embryonic cortical neurons by using shRNA. DYRK1A knockdown compromised neuritogenesis and was associated with alterations in microtubule stability. These experiments demonstrate that MAP1B has DYRK1A-primed and nonprimed GSK3 beta sites that are involved in the regulation of microtubule stability in growing axons.
Scales
Timothy M E
TM
The MRC Centre for Developmental Neurobiology, New Hunt's House, Guy's Campus, King's College London, London SE1 1UL, UK.
Lin
Shen
S
Kraus
Michaela
M
Goold
Robert G
RG
Gordon-Weeks
Phillip R
PR
D000367:Age Factors; D000818:Animals; D001369:Axons / Q000187:drug effects / Q000201:enzymology*; D019556:COS Cells; D002540:Cerebral Cortex / Q000187:drug effects / Q000196:embryology / Q000201:enzymology*; D002522:Chlorocebus aethiops; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D013058:Mass Spectrometry; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000187:drug effects / Q000201:enzymology*; D009154:Mutation; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D051381:Rats; D011993:Recombinant Fusion Proteins; D012694:Serine; D013116:Spinal Cord / Q000196:embryology / Q000201:enzymology; D013912:Threonine; D014162:Transfection; D000097603:Dyrk Kinases
D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D008869:Microtubule-Associated Proteins; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D012694:Serine; D013912:Threonine; D000097603:Dyrk Kinases; C085284:microtubule-associated protein 1B