The MAP kinase pathway is upstream of the activation of GSK3β that enables it to phosphorylate MAP1B and contributes to the stimulation of axon growth

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Abstract

In pheochromocytoma 12 (PC12) cells and sympathetic neurons, nerve growth factor (NGF) engagement with the tropomyosin-related tyrosine kinase (TrkA) receptor activates the serine/threonine kinase glycogen synthase kinase 3β (GSK3β), enabling it to phosphorylate the microtubule-associated protein 1B (MAP1B). GSK3β phosphorylation of MAP1B acts as a molecular switch to regulate microtubule dynamics in growing axons, and hence the rate of axon growth. An important question relates to the identification of the upstream pathway linking the activation of GSK3β with TrkA engagement. TrkA can utilise a number of intracellular signalling pathways, including the mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol-3 kinase (PI3K) pathway. We now show, using pharmacological inhibitor studies of PC12 cells and sympathetic neurons in culture and in vitro kinase and activation assays, that the MAPK pathway, and not the PI3K pathway, links NGF engagement with the TrkA receptor to GSK3β activation in PC12 cells and sympathetic neurons. We also show that activated GSK3β is a small fraction of the total GSK3β present in developing brain and that it is not part of a multiprotein complex. Thus, NGF drives increased neurite growth rates partly by coupling the MAPK pathway to the activation of GSK3β and thereby phosphorylation of MAP1B.

Introduction

A critical event in the differentiation of neurons during embryogenesis is the initiation and extension of cellular processes (neurites) that will eventually form axons and dendrites. Observations of dissociated embryonic neurons in culture have revealed that neurons initially extend several equipotential neurites and that usually only one of these differentiates into an axon, an event that is associated with an increase in growth rate; subsequently, after a variable delay, the remaining neurites become dendrites (Dehmelt et al., 2003, Dotti et al., 1988). During embryogenesis, axon growth rate is regulated by a number of extracellular factors, including members of the neurotrophin family. Neurotrophins enhance axon growth and axon branching (Diamond et al., 1992, Letourneau, 1978, Tucker et al., 2001; reviewed in Tucker, 2002). The archetypal neurotrophin, NGF, is required for the proper innervation of sympathetic neuron targets in the peripheral nervous system (Glebova and Ginty, 2004) and can act directly on axons and growth cones to enhance growth and can even reorient growth cones when applied locally in vitro (Campenot, 1977, Gallo et al., 1997, Grabham and Goldberg, 1997, Gundersen and Barrett, 1979, Letourneau, 1978). We have recently shown that NGF drives enhanced neurite growth rates in PC12 cells and in the axons of cultured superior cervical ganglion (SCG) and dorsal root ganglion neurons by activating the serine/threonine kinase glycogen synthase kinase 3β (GSK3β; Goold and Gordon-Weeks, 2001, Goold and Gordon-Weeks, 2003, Owen and Gordon-Weeks, 2003; reviewed in Gordon-Weeks, 2004).

GSK3β phosphorylates the microtubule-associated protein 1B (MAP1B) in growing axons of differentiating neurons (Goold et al., 1999, Hall et al., 2002, Lucas et al., 1998, Owen and Gordon-Weeks, 2003) and this acts as a molecular switch to control the regulation of microtubule dynamics by MAP1B (Ciani et al., 2004, Goold et al., 1999, Hall et al., 2000, Owen and Gordon-Weeks, 2003), which is essential for the enhancement of axon growth (Gordon-Weeks, 2004) and in the maintenance of the leading extension of migrating cortical neurons (Kawauchi et al., 2003). GSK3β phosphorylation of MAP1B may also be important for growth cone pathfinding (Del Rio et al., 2004). Unactivated GSK3β phosphorylates MAP1B relatively poorly and although NGF can activate GSK3β in certain neuronal cell lines and primary sensory and sympathetic neurons, the mechanism of activation is unknown (Goold and Gordon-Weeks, 2001, Goold and Gordon-Weeks, 2003).

NGF binds either to the TrkA tyrosine kinase receptor or to the p75NTR receptor (Kaplan and Miller, 2000) and it is engagement with the former that is responsible for the activation of GSK3β by NGF (Goold and Gordon-Weeks, 2003) and for the localised effects of NGF on growth cone turning (Gallo et al., 1997). TrkA receptors signal through several, distinct intracellular signalling pathways including the MAPK and PI3K pathways (Kaplan and Miller, 2000), and we show here that it is the MAPK pathway, and not the PI3K pathway, that couples NGF engagement with the TrkA receptor to the activation of GSK3β, and therefore the subsequent phosphorylation of MAP1B. This finding identifies a long sought after link between MAPK activity and the cytoskeleton, and hence axon growth. We also show that only a small fraction of the total GSK3β present in neonatal brain is activated and capable of efficiently phosphorylating MAP1B and that this activation is associated not with participation in a multiprotein complex but with a novel post-translational modification of GSK3β, catalysed by downstream components of the MAPK pathway.

Section snippets

Pharmacological inhibition studies show that the MAPK pathway, but not the PI3K pathway, is upstream of GSK3β activation

We have previously shown that NGF activates GSK3β in PC12 cells and cultured SCG neurons through engagement with the TrkA receptor and not through the p75NTR receptor (Goold and Gordon-Weeks, 2003). NGF engagement with the TrkA receptor can activate a number of intracellular signalling pathways, including the PI3K and MAPK pathways (Kaplan and Miller, 2000). To identify which pathway was responsible, we used specific pathway inhibitors (Davies et al., 2000). When we treated differentiating PC12

The MAPK pathway is upstream of GSK3β activation that enables it to phosphorylate MAP1B

In the experiments reported here, we provide evidence for a novel link between the MAPK pathway and the activation of GSK3β that enables it to phosphorylate MAP1B in PC12 cells and sympathetic neurons in culture, and thereby regulate microtubule dynamics in growing axons and growth cones (Gordon-Weeks, 2004). We have shown, using pharmacological inhibitor studies and in vitro kinase and activation assays, that the MAPK pathway activates GSK3β so that it is able to phosphorylate MAP1B. This

Materials

Dulbecco's Modified Eagle Medium (DMEM), Neurobasal medium, horse serum and foetal bovine serum were purchased from Gibco BRL, Rockville, MD, USA. LY294002, PD98059, UO126 and Wortmannin were purchased from Calbiochem, San Diego, CA, USA. NGF (7.0S) was purchased from Alomone laboratories, Jerusalem, Israel, and recombinant ERK 2 from Calbiochem. All other chemicals were purchased from Sigma, St. Louis, MO, USA.

Cell culture

PC12 cells were maintained on collagen-coated flasks as described previously (Goold

Acknowledgments

We are grateful to The Wellcome Trust for supporting this work. We also thank GlaxoSmithKline for baculovirus expressed GSK3β and Julia Nash for help with the superior cervical ganglion explant cultures. We thank Drs Neil Chadborn, Uwe Drescher and Britta Eickholt and members of our lab for critically reading the manuscript.

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