The Drosophila microtubule associated protein Futsch is phosphorylated by Shaggy/Zeste-white 3 at an homologous GSK3β phosphorylation site in MAP1B
Introduction
During all stages of development, neurons are able to respond plastically to stimuli and can change their morphology accordingly. This ability is mostly based on the dynamic rearrangement of actin filaments and microtubules (Dent and Gertler, 2003, Kalil and Dent, 2005). The regulation of microtubule dynamics depends on microtubule-associated proteins (MAPs). MAP1B is the earliest MAP to be expressed in developing neurons and although the precise function of MAP1B is unclear, a role in axonal extension and growth cone function in developing and regenerating neurons has been inferred from a variety of experiments (Gordon-Weeks and Fischer, 2000, Gonzalez-Billault et al., 2004). These include the depletion of MAP1B by antisense RNA and the derivation of transgenic mice (DiTella et al., 1996, Takei et al., 1997, Takei et al., 2000, Meixner et al., 2000, Gonzalez-Billault et al., 2001, Gonzalez-Billault et al., 2002, Teng et al., 2001). The precise function of MAP1B may depend on its particular phosphorylation state and a number of kinases have been implicated in phosphorylating MAP1B including glycogen synthase kinase 3β (GSK3β), cyclin-dependent kinase 5, casein kinase 2 and c-Jun N-terminal kinase (Díaz-Nido et al., 1988, Lucas et al., 1998, Goold and Gordon-Weeks, 2001, Goold et al., 1999, Kawauchi et al., 2003, Kawauchi et al., 2005). A local role for phosphorylated MAP1B in growth cone turning has been revealed by microscale chromophore-assisted laser inactivation (Mack et al., 2000). Furthermore, axonal guidance cues, such as netrins, have been shown to stimulate GSK3β activity and MAP1B phosphorylation (Del Rio et al., 2004).
We recently identified the Drosophila MAP1B homologue, Futsch, as a large protein (> 570 kDa) that associates with microtubules (Hummel et al., 2000). Both, the N- and C-terminal domains are homologous to vertebrate MAP1B. The central domain of Futsch, however, is highly repetitive with a 37 amino acid motif repeated 66 times. These repeats are homologous to similar repeats in vertebrate neurofilament proteins and show little homology to any known vertebrate MAPs. futsch mutants were identified on the basis of an absence of the monoclonal antibody (mAb) 22C10 epitope, which is located in the Futsch protein (Hummel et al., 2000). Subsequent analysis revealed a function for Futsch during axonal growth as well as synapse formation in Drosophila (Roos et al., 2000, Ruiz-Canada et al., 2004) and in neuronal maintenance in adult flies (Bettencourt et al., 2005).
To determine the extent of functional homology between Futsch and MAP1B, we conducted an analysis of the most conserved N- and C-terminal protein domains. In common with MAP1B, Futsch is phosphorylated in a spatially and temporally regulated, GSK3β-dependent manner. MAP1B function during microtubule dynamics is directly regulated by GSK3β (Lucas et al., 1998, Goold et al., 1999, Goold and Gordon-Weeks, 2001, Owen and Gordon-Weeks, 2003). Here we show that an antibody generated against one conserved GSK3β phosphorylation site in MAP1B (Trivedi et al., 2005) cross-reacts with Drosophila Futsch. The tissue distribution of this epitope during Drosophila development is similar, but not identical, to the total Futsch protein pool, as described for MAP1B (Trivedi et al., 2005). Additionally, we provide biochemical evidence that Shaggy/Zeste-white 3, the Drosophila GSK3 homologue, can directly phosphorylate Futsch in vitro at this conserved site.
Section snippets
Characterization of the Futsch protein
Western blot analysis reveals that the Drosophila Futsch protein has a molecular weight of more than 570 kDa (Hummel et al., 2000). The most recent annotation of the Drosophila genome (Release 4.2.1) shows that the corresponding gene is spread over 8 exons. As we have previously shown, the 5′ exon encodes a 450 amino acid domain that is homologous to the N-terminal domain of vertebrate MAP1B, whereas the last predicted exon encodes a 250 amino acid stretch homologous to the C-terminus of MAP1B (
Discussion
Here we have analyzed the Drosophila Futsch protein whose closest vertebrate homologue is the large microtubule associated protein MAP1B (Hummel et al., 2000). Annotation of the Drosophila genome sequence predicts a large gene encoding a putative protein with more than 5400 amino acids. The futsch gene product was originally characterized as the epitope for mAb 22C10 and this antibody recognizes a protein of over 500 kDa by Western blotting. Futsch and MAP1B share conserved N- and C-terminal
Fly strains
All fly crosses were performed on standard food at 25°C. The following Drosophila strains were used: w118 (wild type); futschK68/futschK68, futschN94/futschN94 (Hummel et al., 2000); sggK22 (Bourouis et al., 1989); wingless1 (Couso and Martinez, 1994); DF(1)AC7 (1E3-4; 2A), 22C10 negative; EP(X)1419; elav-GAL4, da-GAL4 and ftz-GAL4 (stocks provided by Bloomington Stock Center, University of Indiana, USA); UAS-sgg, UAS-sggactivated, UAS-sggDN (obtained from M. Bourouis, Université de Nice, Nice,
Acknowledgments
We thank Erich Buchner for mAb nc46 (Reichmuth et al., 1995), Marc Bourouis for mAb 2G2C5 (Ruel et al., 1993b) and the following fly strains: UAS-sgg, UAS-sggactivated, UAS-sggDN. We are particularly grateful to Shen Lin for the expression of the C-terminal fusion protein of Futsch (amino acids 5201–5347). This work was supported through funding of the DFG to C.K. and the MRC to P.G-W.
References (50)
- et al.
Notch is required for wingless signaling in the epidermis of Drosophila
Cell
(1994) - et al.
MAP1B is required for Netrin 1 signaling in neuronal migration and axonal guidance
Curr. Biol.
(2004) - et al.
Cytoskeletal dynamics and transport in growth cone motility and axon guidance
Neuron
(2003) - et al.
Microtubule-associated protein 1B is involved in the initial stages of axonogenesis in peripheral nervous system cultured neurons
Brain Res.
(2002) - et al.
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
Mol. Cell. Neurosci.
(2005) - et al.
Axonal remodeling and synaptic differentiation in the cerebellum is regulated by WNT-7a signaling
Cell
(2000) - et al.
MAP1B is encoded as a polyprotein that is processed to form a complex N-terminal microtubule-binding domain
Neuron
(1991) - et al.
Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension
Gene
(1989) - et al.
Commissure formation in the embryonic CNS of Drosophila
Dev. Biol.
(1999) - et al.
Drosophila Futsch/22C10 is a MAP1B-like protein required for dendritic and axonal development
Neuron
(2000)
Touch and go: guidance cues signal to the growth cone cytoskeleton
Curr. Opin. Neurobiol.
MAP1B phosphorylation is differentially regulated by Cdk5/p35, Cdk5/p25, and JNK
Biochem. Biophys. Res. Commun.
The microtubule-associated protein MAP1B is involved in local stabilization of turning growth cones
Mol. Cell. Neurosci.
Inhibition of glycogen synthase kinase 3β in sensory neurons in culture alters filopodia dynamics and microtubule distribution in growth cones
Mol. Cell. Neurosci.
The Drosophila Wnt, wingless, provides an essential signal for pre- and postsynaptic differentiation
Cell
The sap47 gene of Drosophila melanogaster codes for a novel conserved neuronal protein associated with synaptic terminals
Brain Res. Mol. Brain Res.
Drosophila Futsch regulates synaptic microtubule organization and is necessary for synaptic growth
Neuron
New synaptic bouton formation is disrupted by misregulation of microtubule stability in aPKC mutants
Neuron
Genetic dissection of structural and functional components of synaptic plasticity: I. Fasciclin II controls synaptic stabilization and growth
Neuron
A 45 amino acid residue domain necessary and sufficient for proteolytic cleavage of the MAP1B polyprotein precursor
FEBS Lett.
Disruption of the MAP1B-related protein FUTSCH leads to changes in the neuronal cytoskeleton, axonal transport defects, and progressive neurodegeneration in Drosophila
Mol. Biol. Cell
Shaggy/GSK-3β kinase localizes to the centrosome and to specialized cytoskeletal structures in Drosophila
Cell Motil. Cytoskeleton
Targeted increase in shaggy activity levels blocks wingless signaling
Genesis
Mutant Drosophila embryos in which all cells adopt a neural fate
Nature
Targeted gene expression as a means of altering cell fates and generating dominant phenotypes
Development
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