The drebrin/EB3 pathway regulates cytoskeletal dynamics to drive neuritogenesis in embryonic cortical neurons.

Co-ordinating the dynamic behaviour of actin filaments (F-actin) and microtubules in filopodia is an important underlying process in neuritogenesis, but the molecular pathways involved are ill-defined. The drebrin/end-binding protein 3 (EB3) pathway is a candidate pathway for linking F-actin to microtubules in filopodia. Drebrin binds F-actin and, simultaneously, the microtubule-binding protein EB3 when bound to microtubule plus-ends. We assessed the effect on neuritogenesis of gain- or loss-of-function of proteins in the drebrin/EB3 pathway in rat embryonic cortical neurons in culture. Loss-of-function of drebrin by gene editing or pharmacological inhibition of drebrin binding to F-actin reduced the number of dynamic microtubules in the cell periphery and simultaneously delayed the initiation of neuritogenesis, whereas over-expression of drebrin induced supernumerary neurites. Similarly, loss of EB3 inhibited neuritogenesis, whereas loss of end-binding protein 1 (EB1), a related protein that does not bind to drebrin, did not affect neuritogenesis. Over-expression of EB3, but not EB1, induced supernumerary neurites. We discovered that EB3 is more proximally located at dynamic microtubule plus-ends than EB1 in growth cone filopodia allowing for continuous microtubule elongation as the drebrin/EB3 pathway zippers microtubules to F-actin in filopodia. Finally, we showed that preventing the entry of dynamic microtubules into filopodia using a pharmacological inhibitor of microtubule dynamics is associated with a loss of EB3, but not EB1, from microtubule plus-ends and a concurrent attenuation of neuritogenesis. Collectively, these findings support the idea that neuritogenesis depends on microtubule/F-actin zippering in filopodia orchestrated by the drebrin/EB3 pathway.

Phosphorylation of Drebrin and Its Role in Neuritogenesis.

Neuritogenesis is an early event in neuronal development in which newborn neurons first form growth cones, as a prerequisite for the formation of axons and dendrites. Growth cones emerge from segmented regions of the lamellipodium of embryonic neurons and grow away from the cell body leaving behind a neurite that will eventually polarise into an axon or dendrite. Growth cones also function to navigate precise routes through the embryo to locate an appropriate synaptic partner. Dynamic interactions between two components of the neuronal cytoskeleton, actin filaments and microtubules, are known to be essential for growth cone formation and hence neuritogenesis. The molecular mechanisms that coordinate interactions between actin filaments and dynamic microtubules during neuritogenesis are beginning to be understood. One candidate pathway coupling actin filaments to microtubules consists of the actin filament-binding protein drebrin and the microtubule-binding +TIP protein EB3. This pathway is regulated proximally by cyclin-dependent kinase 5 phosphorylation of drebrin but the upstream elements in the pathway have yet to be identified.

The Role of Drebrin in Cancer Cell Invasion.

Cancer progression is characterized by the capacity of malignant cells to exploit an innate migratory ability in order to invade adjacent tissues, enter the vasculature and eventually metastasize to secondary organs. It is this spread of cancer cells that is the major cause of death in cancer patients. Understanding the basic biology of how cancer cells generate an invasive phenotype will be crucial to the identification of drug targets with the aim of impeding tumour dissemination. Ten years on from its initial description in neuronal cells, drebrin expression was found in a wide variety of non-neuronal cells that importantly included cancer cell lines. Since then mounting evidence suggests that drebrin may be a key player in the advancement of several diverse cancer types where its expression is frequently upregulated. Cancer cell motility and invasion are crucial elements in the metastatic cascade and involve dramatic changes in cellular morphology that are associated with dynamic remodelling of the cytoskeleton. Interestingly, it now appears that drebrin could deliver this role during cancer development.

Drebrin-mediated microtubule-actomyosin coupling steers cerebellar granule neuron nucleokinesis and migration pathway selection.

Neuronal migration from a germinal zone to a final laminar position is essential for the morphogenesis of neuronal circuits. While it is hypothesized that microtubule-actomyosin crosstalk is required for a neuron's 'two-stroke' nucleokinesis cycle, the molecular mechanisms controlling such crosstalk are not defined. By using the drebrin microtubule-actin crosslinking protein as an entry point into the cerebellar granule neuron system in combination with super-resolution microscopy, we investigate how these cytoskeletal systems interface during migration. Lattice light-sheet and structured illumination microscopy reveal a proximal leading process nanoscale architecture wherein f-actin and drebrin intervene between microtubules and the plasma membrane. Functional perturbations of drebrin demonstrate that proximal leading process microtubule-actomyosin coupling steers the direction of centrosome and somal migration, as well as the switch from tangential to radial migration. Finally, the Siah2 E3 ubiquitin ligase antagonizes drebrin function, suggesting a model for control of the microtubule-actomyosin interfaces during neuronal differentiation.

The role of the drebrin/EB3/Cdk5 pathway in dendritic spine plasticity, implications for Alzheimer's disease.

The drebrin/EB3/Cdk5 intracellular signalling pathway couples actin filaments to dynamic microtubules in cellular settings where cells are changing shape. The pathway has been most intensively studied in neuronal development, particularly neuritogenesis and neuronal migration, and in synaptic plasticity at dendritic spines in mature neurons. Drebrin is an actin filament side-binding and bundling protein that stabilises actin filaments. The end-binding (EB) proteins are microtubule plus-end tracking proteins (+TIPs) that localise to the growing plus-ends of dynamic microtubules and regulate their behavior and the binding of other +TIP proteins. EB3 binds specifically to drebrin when drebrin is bound to actin filaments, for example at the base of a growth cone filopodium, and EB3 is located at the plus-end of a growing microtubule inserting into the filopodium. This interaction therefore forms the basis for coupling dynamic microtubules to actin filaments in growth cones of developing neurons. Appropriate responses to growth cone guidance cues depend on actin filament/microtubule co-ordination in the growth cone, although the role of the drebrin/EB3/Cdk5 pathway in this context has not been directly tested. A similar cytoskeleton coupling pathway operates in dendritic spines in mature neurons where the activity-dependent insertion of dynamic microtubules into dendritic spines is facilitated by drebrin binding to EB3. Microtubule insertion into dendritic spines drives spine maturation during long-term potentiation and therefore has a role in synaptic plasticity and memory formation. In Alzheimer's disease and related chronic neurodegenerative diseases, there is an early and dramatic loss of drebrin from dendritic spines that precedes synapse loss and neurodegeneration and might contribute to a failure of synaptic plasticity and hence to cognitive decline.

The Actin-Binding Protein Drebrin Inhibits Neointimal Hyperplasia.

OBJECTIVE: Vascular smooth muscle cell (SMC) migration is regulated by cytoskeletal remodeling as well as by certain transient receptor potential (TRP) channels, nonselective cation channels that modulate calcium influx. Proper function of multiple subfamily C TRP (TRPC) channels requires the scaffolding protein Homer 1, which associates with the actin-binding protein Drebrin. We found that SMC Drebrin expression is upregulated in atherosclerosis and in response to injury and investigated whether Drebrin inhibits SMC activation, either through regulation of TRP channel function via Homer or through a direct effect on the actin cytoskeleton. APPROACH AND RESULTS: Wild-type (WT) and congenic Dbn(-/+) mice were subjected to wire-mediated carotid endothelial denudation. Subsequent neointimal hyperplasia was 2.4±0.3-fold greater in Dbn(-/+) than in WT mice. Levels of globular actin were equivalent in Dbn(-/+) and WT SMCs, but there was a 2.4±0.5-fold decrease in filamentous actin in Dbn(-/+) SMCs compared with WT. Filamentous actin was restored to WT levels in Dbn(-/+) SMCs by adenoviral-mediated rescue expression of Drebrin. Compared with WT SMCs, Dbn(-/+) SMCs exhibited increased TRP channel activity in response to platelet-derived growth factor, increased migration assessed in Boyden chambers, and increased proliferation. Enhanced TRP channel activity and migration in Dbn(-/+) SMCs were normalized to WT levels by rescue expression of not only WT Drebrin but also a mutant Drebrin isoform that binds actin but fails to bind Homer. CONCLUSIONS: Drebrin reduces SMC activation through its interaction with the actin cytoskeleton but independently of its interaction with Homer scaffolds.

Drebrin regulates neuroblast migration in the postnatal mammalian brain.

After birth, stem cells in the subventricular zone (SVZ) generate neuroblasts that migrate along the rostral migratory stream (RMS) to become interneurons in the olfactory bulb (OB). This migration is crucial for the proper integration of newborn neurons in a pre-existing synaptic network and is believed to play a key role in infant human brain development. Many regulators of neuroblast migration have been identified; however, still very little is known about the intracellular molecular mechanisms controlling this process. Here, we have investigated the function of drebrin, an actin-binding protein highly expressed in the RMS of the postnatal mammalian brain. Neuroblast migration was monitored both in culture and in brain slices obtained from electroporated mice by time-lapse spinning disk confocal microscopy. Depletion of drebrin using distinct RNAi approaches in early postnatal mice affects neuroblast morphology and impairs neuroblast migration and orientation in vitro and in vivo. Overexpression of drebrin also impairs migration along the RMS and affects the distribution of neuroblasts at their final destination, the OB. Drebrin phosphorylation on Ser142 by Cyclin-dependent kinase 5 (Cdk5) has been recently shown to regulate F-actin-microtubule coupling in neuronal growth cones. We also investigated the functional significance of this phosphorylation in RMS neuroblasts using in vivo postnatal electroporation of phosphomimetic (S142D) or non-phosphorylatable (S142A) drebrin in the SVZ of mouse pups. Preventing or mimicking phosphorylation of S142 in vivo caused similar effects on neuroblast dynamics, leading to aberrant neuroblast branching. We conclude that drebrin is necessary for efficient migration of SVZ-derived neuroblasts and propose that regulated phosphorylation of drebrin on S142 maintains leading process stability for polarized migration along the RMS, thus ensuring proper neurogenesis.

Neuronal cytoskeleton in synaptic plasticity and regeneration.

During development, dynamic changes in the axonal growth cone and dendrite are necessary for exploratory movements underlying initial axo-dendritic contact and ultimately the formation of a functional synapse. In the adult central nervous system, an impressive degree of plasticity is retained through morphological and molecular rearrangements in the pre- and post-synaptic compartments that underlie the strengthening or weakening of synaptic pathways. Plasticity is regulated by the interplay of permissive and inhibitory extracellular cues, which signal through receptors at the synapse to regulate the closure of critical periods of developmental plasticity as well as by acute changes in plasticity in response to experience and activity in the adult. The molecular underpinnings of synaptic plasticity are actively studied and it is clear that the cytoskeleton is a key substrate for many cues that affect plasticity. Many of the cues that restrict synaptic plasticity exhibit residual activity in the injured adult CNS and restrict regenerative growth by targeting the cytoskeleton. Here, we review some of the latest insights into how cytoskeletal remodeling affects neuronal plasticity and discuss how the cytoskeleton is being targeted in an effort to promote plasticity and repair following traumatic injury in the central nervous system.

Drebrin contains a cryptic F-actin-bundling activity regulated by Cdk5 phosphorylation.

Drebrin is an actin filament (F-actin)-binding protein with crucial roles in neuritogenesis and synaptic plasticity. Drebrin couples dynamic microtubules to F-actin in growth cone filopodia via binding to the microtubule-binding +TIP protein EB3 and organizes F-actin in dendritic spines. Precisely how drebrin interacts with F-actin and how this is regulated is unknown. We used cellular and in vitro assays with a library of drebrin deletion constructs to map F-actin binding sites. We discovered two domains in the N-terminal half of drebrin-a coiled-coil domain and a helical domain-that independently bound to F-actin and cooperatively bundled F-actin. However, this activity was repressed by an intramolecular interaction relieved by Cdk5 phosphorylation of serine 142 located in the coiled-coil domain. Phospho-mimetic and phospho-dead mutants of serine 142 interfered with neuritogenesis and coupling of microtubules to F-actin in growth cone filopodia. These findings show that drebrin contains a cryptic F-actin-bundling activity regulated by phosphorylation and provide a mechanistic model for microtubule-F-actin coupling.

MAP1B enhances microtubule assembly rates and axon extension rates in developing neurons.

The microtubule-associated protein MAP1B is known to have important roles in neuronal development, particularly during neuronal migration and axonogenesis, but its precise molecular actions are unknown. We used RNA interference silencing of protein expression to specifically knock down MAP1B in cultured embryonic rat cortical neurons. Reduction of MAP1B in these neurons is associated with several abnormal morphological phenotypes including the production of more highly branched and slower growing axons than normal. MAP1B binds to dynamic microtubules and indirect evidence suggests that MAP1B regulates microtubule dynamics. We used the +TIP protein EB3 to assess the dynamic behaviour and orientation of microtubules in neurons in which MAP1B had been knocked down. This revealed a reduction in the speed of microtubule growth in the proximal and distal axon shaft, but not in growth cone filopodia. These observations suggest that the function of MAP1B is to suppress axon branching and enhance axon growth and that this is achieved by maintaining dynamic microtubule growth. To test this hypothesis we expressed MAP1B in a cell line that does not have endogenous MAP1B, this led to an increase in microtubule elongation rates. These findings show that MAP1B enhances microtubule assembly rates and axon extension rates in developing neurons by binding to dynamic microtubules.

Evidence that glycogen synthase kinase-3 isoforms have distinct substrate preference in the brain.

Mammalian glycogen synthase kinase-3 (GSK3) is generated from two genes, GSK3α and GSK3ß, while a splice variant of GSK3ß (GSK3ß2), containing a 13 amino acid insert, is enriched in neurons. GSK3α and GSK3ß deletions generate distinct phenotypes. Here, we show that phosphorylation of CRMP2, CRMP4, ß-catenin, c-Myc, c-Jun and some residues on tau associated with Alzheimer's disease, is altered in cortical tissue lacking both isoforms of GSK3. This confirms that they are physiological targets for GSK3. However, deletion of each GSK3 isoform produces distinct substrate phosphorylation, indicating that each has a different spectrum of substrates (e.g. phosphorylation of Thr509, Thr514 and Ser518 of CRMP is not detectable in cortex lacking GSK3ß, yet normal in cortex lacking GSK3α). Furthermore, the neuron-enriched GSK3ß2 variant phosphorylates phospho-glycogen synthase 2 peptide, CRMP2 (Thr509/514), CRMP4 (Thr509), Inhibitor-2 (Thr72) and tau (Ser396), at a lower rate than GSK3ß1. In contrast phosphorylation of c-Myc and c-Jun is equivalent for each GSK3ß isoform, providing evidence that differential substrate phosphorylation is achieved through alterations in expression and splicing of the GSK3 gene. Finally, each GSK3ß splice variant is phosphorylated to a similar extent at the regulatory sites, Ser9 and Tyr216, and exhibit identical sensitivities to the ATP competitive inhibitor CT99021, suggesting upstream regulation and ATP binding properties of GSK3ß1 and GSK3ß2 are similar.

Evolution of the spatial distribution of MAP1B phosphorylation sites in vertebrate neurons.

The microtubule-associated protein MAP1B has important roles in neural development, particularly in migrating and differentiating neurons. MAP1B is phosphorylated by glycogen synthase kinase 3beta (GSK-3beta) at a site that requires prior phosphorylation by another kinase four amino acid residues downstream of the GSK-3beta site, a so-called primed site, and at non-primed sites that have no such requirement. In developing mammalian neurons, MAP1B phosphorylated by GSK-3beta at primed and non-primed sites is distributed in spatially distinct patterns. Non-primed GSK-3beta-phosphorylated MAP1B sites are only expressed in axons and are present in the form of a gradient that is highest distally, towards the growth cone. In contrast, primed GSK-3beta-phosphorylated MAP1B sites are present throughout the neuron including the somato-dendritic compartment and uniformly throughout the axon. To examine the function of these two sites, we explored the evolutionary conservation of the spatial distribution of GSK-3beta primed and non-primed sites on MAP1B in vertebrate neurons. We immunostained spinal cord sections from embryonic or newly hatched representatives of all of the main vertebrate groups using phospho-specific antibodies to GSK-3beta primed and non-primed sites on MAP1B. This revealed a remarkable evolutionary conservation of the distribution of primed and non-primed GSK-3beta-phosphorylated MAP1B sites in developing vertebrate neurons. By analysing amino acid sequences of MAP1B we found that non-primed GSK-3beta sites are more highly conserved than primed sites throughout the vertebrates, suggesting that the latter evolved later. Finally, distinct distribution patterns of GSK-3beta primed and non-primed sites on MAP1B were preserved in cultured rat embryonic cortical neurons, opening up the possibility of studying the two sites in vitro.

The neuron-specific isoform of glycogen synthase kinase-3beta is required for axon growth.

Glycogen synthase kinase-3 (GSK-3) has become an important target for the treatment of mood disorders and neurodegenerative disease. It comprises three enzymes, GSK-3alpha, beta and the neuron-specific isoform, beta2. GSK-3 regulates axon growth by phosphorylating microtubule-associated proteins including Tau. A genetic polymorphism that leads to an increase in the ratio of GSK-3beta1 to GSK-3beta2 interacts with Tau haplotypes to modify disease risk in Parkinson's and Alzheimer's disease. We have examined the roles of each isoform of GSK-3 in neurons. Silencing of GSK-3beta2 inhibited retinoic acid-induced neurite outgrowth in SH-SY5Y neuroblastoma cells and axon growth in rat cortical neurons. Inhibition of neurite outgrowth was prevented by co-expression of GSK-3beta2 but not by co-expression of GSK-3alpha or GSK-3beta1. Ectopic expression GSK-3beta2 enhanced the effects of retinoic acid on neurite length and induced neurite formation in the absence of retinoic acid. GSK-3beta2 phosphorylated Tau at a subset of those sites phosphorylated by GSK-3beta1. In addition, Axin, which regulates responses to Wnt signals, associated more readily with GSK-3beta1 than with GSK-3beta2. Our results suggest that GSK-3 inhibitors that target the Axin-binding site in GSK-3 will preserve the beneficial effects of GSK-3beta2 on axon growth.

Cytoskeletal dynamics in growth-cone steering.

Interactions between dynamic microtubules and actin filaments are essential to a wide range of cell biological processes including cell division, motility and morphogenesis. In neuronal growth cones, interactions between microtubules and actin filaments in filopodia are necessary for growth cones to make a turn. Growth-cone turning is a fundamental behaviour during axon guidance, as correct navigation of the growth cone through the embryo is required for it to locate an appropriate synaptic partner. Microtubule-actin filament interactions also occur in the transition zone and central domain of the growth cone, where actin arcs exert compressive forces to corral microtubules into the core of the growth cone and thereby facilitate microtubule bundling, a requirement for axon formation. We now have a fairly comprehensive understanding of the dynamic behaviour of the cytoskeleton in growth cones, and the stage is set for discovering the molecular machinery that enables microtubule-actin filament coupling in growth cones, as well as the intracellular signalling pathways that regulate these interactions. Furthermore, recent experiments suggest that microtubule-actin filament interactions might also be important for the formation of dendritic spines from filopodia in mature neurons. Therefore, the mechanisms coupling microtubules to actin filaments in growth-cone turning and dendritic-spine maturation might be conserved.

An alternatively spliced form of glycogen synthase kinase-3beta is targeted to growing neurites and growth cones.

The serine/threonine kinase glycogen synthase kinase-3beta (GSK-3beta) is expressed in two, alternatively spliced, isoforms: a short form (GSK-3beta1) and a long form containing a 13 amino acid insert in the catalytic domain (GSK-3beta2). We examined the expression of these isoforms in the rat using specific antibodies and found that GSK-3beta2, in contrast to GSK-3beta1, is only expressed in the nervous system. The highest levels of GSK-3beta2 are found in the developing nervous system but expression persists into adulthood. In the adult central nervous system the highest expression of GSK-3beta2 occurs in regions with a high proportion of white matter, suggesting that GSK-3beta2 is expressed in axons. Consistent with this finding, sub-cellular fractionation of neonatal rat brain showed that GSK-3beta2 is present in fractions enriched in neurites and growth cones. Furthermore, we found that when we separated neuronal cell bodies from neurites by culturing embryonic cortical neurons in neurite outgrowth inserts, GSK-3beta2 was present in both compartments. Finally, a rabbit polyclonal antibody raised to the 13 amino acid insert of GSK-3beta2 (anti-8A) that specifically recognises GSK-3beta2, labels the cell body, including the nucleus, neurites and growth cones of embryonic neurons in culture. To compare functionally the two isoforms, we performed in vitro kinase assays. These showed that GSK-3beta1 is more efficient at phosphorylating the microtubule-associated protein MAP1B than GSK-3beta2, consistent with previous findings with the microtubule-associated protein tau. However, when co-expressed with MAP1B in COS-7 cells, both GSK-3beta isoforms equally efficiently phosphorylated MAP1B and had a similar influence on the regulation of microtubule dynamics by MAP1B in these cells. We conclude that the alternatively spliced isoform of GSK-3beta, GSK-3beta2, is neuron-specific and has overlapping activities with GSK-3beta1.

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.

Targeting of the F-actin-binding protein drebrin by the microtubule plus-tip protein EB3 is required for neuritogenesis.

Interactions between dynamic microtubules and actin filaments (F-actin) underlie a range of cellular processes including cell polarity and motility. In growth cones, dynamic microtubules are continually extending into selected filopodia, aligning alongside the proximal ends of the F-actin bundles. This interaction is essential for neuritogenesis and growth-cone pathfinding. However, the molecular components mediating the interaction between microtubules and filopodial F-actin have yet to be determined. Here we show that drebrin, an F-actin-associated protein, binds directly to the microtubule-binding protein EB3. In growth cones, this interaction occurs specifically when drebrin is located on F-actin in the proximal region of filopodia and when EB3 is located at the tips of microtubules invading filopodia. When this interaction is disrupted, the formation of growth cones and the extension of neurites are impaired. We conclude that drebrin targets EB3 to coordinate F-actin-microtubule interactions that underlie neuritogenesis.

The immunolocalization of the synaptic glycoprotein neuroplastin differs substantially between the human and the rodent brain.

Neuroplastin is a cell adhesion molecule of the immunoglobulin superfamily that exists in two splice isoforms, np65/np55, and that was reported to play a prominent role in synaptic plasticity processes. The splice isoform np65 associates with synapses in an activity-dependent manner and has been shown to play a role for the induction of hippocampal long-term potentiation in rodents. We have therefore analyzed the distribution of neuroplastins in human brain. Neuroplastin is present in many neuronal cell types of the forebrain and cerebellum and immunoreactive label covers the cell soma, neurites and also puncta in the neuropil were visible. Interestingly, we found some remarkable species differences in the expression patterns of neuroplastins between the human and the rodent brain. In human brain np65 is prominently present in cerebellum while np55 is the predominant isoform in mouse and rat cerebellum. Moreover, the parasagittal stripe-type of staining seen with np55 in mouse cerebellum is not found in human brain. In addition we found no segregation of np65 immunolabel in hippocampal subregions like it was reported previously for the rat. These results might indicate different cellular functions of the molecule in different species.

The Drosophila microtubule associated protein Futsch is phosphorylated by Shaggy/Zeste-white 3 at an homologous GSK3beta phosphorylation site in MAP1B.

The Drosophila homologue of the microtubule associated protein MAP1B is encoded by the futsch locus. The deduced protein Futsch is about twice the size of MAP1B and shows high homology in the N- and C-terminal domains. The central part of Futsch is characterized by a highly repetitive structure based on a 37 amino acid motif. Futsch, like MAP1B, colocalizes with microtubules and is necessary for the organization of the microtubule cytoskeleton during axonal growth and synaptogenesis. To further analyze the functional relevance of Futsch as a MAP1B-like protein, we performed a molecular analysis of the conserved protein domains. Using a number of antisera, we show that, unlike the MAP1B polyprotein, which is cleaved to generate a heavy and light chain, Futsch is expressed as a single protein. The function of MAP1B is in part regulated by phosphorylation mediated by kinases that include casein kinase 2 and glycogen synthase kinase 3beta (GSK3beta). We show here that at least one GSK3beta phosphorylation site of MAP1B is conserved in Futsch and that this site can be phosphorylated by GSK3beta and its Drosophila homologue, Shaggy/Zeste-white 3. To test the functional relevance of these findings we generated a number of minigenes and assayed their ability to rescue the phenotype of futsch mutants. Our data highlight some differences between MAP1B and Futsch but demonstrate that important structural and functional aspects are conserved between fly and vertebrate members of this protein family.

The MAP kinase pathway is upstream of the activation of GSK3beta that enables it to phosphorylate MAP1B and contributes to the stimulation of axon growth.

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 3beta (GSK3beta), enabling it to phosphorylate the microtubule-associated protein 1B (MAP1B). GSK3beta 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 GSK3beta 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 GSK3beta activation in PC12 cells and sympathetic neurons. We also show that activated GSK3beta is a small fraction of the total GSK3beta 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 GSK3beta and thereby phosphorylation of MAP1B.