Glycogen synthase kinase-3β phosphorylation of MAP1B at Ser1260 and Thr1265 is spatially restricted to growing axons

Abstract

Recent experiments show that the microtubule-associated protein (MAP) 1B is a major phosphorylation substrate for the serine/threonine kinase glycogen synthase kinase-3β (GSK-3β) in differentiating neurons. GSK-3β 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-3β 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-3β at Ser1260 and Thr1265 in vivo. We also showed that in the developing nervous system of rat embryos, the expression of GSK-3β phosphorylated MAP1B is spatially restricted to growing axons, in a gradient that is highest distally, despite the expression of MAP1B and GSK-3β throughout the entire neuron. This suggests that there is a mechanism that spatially regulates the GSK-3β 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-3β phosphorylation sites contribute to the regulation of microtubule dynamics by MAP1B.

Sustained GSK3 activity markedly facilitates nerve regeneration

“…20), specifically abrogated increased axonal growth, while lithium, a non-competitive inhibitor impeding GSK3 activity via S21/9 phosphorylation, showed no effect on GSK3 S/A neurons. Together with the observation that direct phosphorylation of MAP1B at Th1265 by GSK3, which does not require prior priming by other kinases 16,22,36 , was significantly elevated in neurons of double knock-in mice, these data reflect an overall higher kinase activity of GSK3 S/A compared with controls. Moreover, these data exclude the inhibition of the GSK3 kinase activity by another, unknown compensatory mechanism in GSK3 S/A mice.…”

Distinct Roles of c-Jun N-Terminal Kinase Isoforms in Neurite Initiation and Elongation during Axonal Regeneration

“…The function of MAP1B in stabilizing axonal microtubules is regulated by phosphorylation through two mechanisms: mode II phosphorylation via casein kinase II is observed until adulthood. Mode I is developmentally regulated and implies PDPKs (proline-directed protein kinases), such as GSK-3␤, Cdk-5, and MAPK (JNKs) (Ulloa et al, 1993;Kawauchi et al, 2003Kawauchi et al, , 2005Trivedi et al, 2005) (for references and review, see Riederer, 2007). Nonphosphorylated and mode II phosphorylated MAP1B is found in all compartments of neuronal cytoplasm.…”

An Essential Role for the Integrin-Linked Kinase–Glycogen Synthase Kinase-3β Pathway during Dendrite Initiation and Growth

“…These might include CaMKII (Miller and Kaplan, 2003) and/or PKC (Wang et al, 2006), the latter of which is known to regulate ERK activity, in part through GSK-3␤ (Wang et al, 2006). In addition to ERK1/2, GSK-3␤ has been reported to regulate axonal growth and transport by modulating the phosphorylation of a number of other targets, including Tau (Morfini et al, 2002), and cytoskeletal proteins such as MAP1B (Trivedi et al, 2005) and MAP2 (Sanchez et al, 1998(Sanchez et al, , 2000. We are currently systematically examining the phosphorylation of each of these proteins in neurons treated with ILK and GSK-3␤ inhibitors.…”