DMAP‐85: A τ‐Like Protein from Drosophila melanogaster Larvae

Abstract

Microtubule‐associated proteins (MAPs) play major regulatory roles in the organization and integrity of the cytoskeletal network. Our main interest in this study was the identification and the analysis of structural and functional aspects of Drosophila melanogaster MAPs. A novel MAP with a relative molecular mass of 85 kDa from Drosophila larvae was found associated with taxol‐polymerized microtubules. In addition, this protein bound to mammalian tubulin in an overlay assay and coassembled with purified bovine brain tubulin in microtubule sedimentation experiments. The estimated stoichiometry of 85‐kDa protein versus tubulin in the polymers was 1:5.3 ± 0.2 mol/mol. It was shown that the 85‐kDa protein bound specifically to an affinity column of Sepharose‐βII‐(422–434) tubulin peptide, which contains the sequence of the MAP binding domain on βII‐tubulin. Affinity‐purified 85‐kDa protein enhanced microtubule assembly in a concentration‐dependent manner. This effect was significantly decreased by the presence of the βII‐(422–434) peptide in the assembly assays, thus confirming the specificity of the 85‐kDa protein interaction with the C‐terminal domain on tubulin. Furthermore, this protein also exhibited a strong affinity for calmodulin, based on affinity chromatographic assays. Monoclonal and polyclonal anti‐τ antibodies, including sequence‐specific probes that recognize repeated microtubule‐binding motifs on τ, MAP‐2, and MAP‐4 and specific N‐terminal sequences of τ, cross‐reacted with the 85‐kDa protein from Drosophila larvae. These results suggest that τ and Drosophila 85‐kDa protein share common functional and structural epitopes. We have named this protein as DMAP‐85 for Drosophila MAP. The finding on a Drosophila protein with functional homology and structural similarities to mammalian τ opens new perspectives to understand the cellular roles of MAPs.

Neuroinflammation in the pathogenesis of Alzheimer’s disease. A rational framework for the search of novel therapeutic approaches

“…This process of neurodegeneration self amplifies when τ aggregates are released into the extracellular environment, since there is an important body of evidence supporting neurotoxicity of τ aggregates (Neumann et al, 2011). Among that evidence we can mention cell culture studies showing that overexpression of τ alters cell morphology, delays cell growth, and changes the distribution of organelles that are transported through the axis by motor axonal microtubule-associated proteins (reviewed in Cambiazo et al, 1995). Moreover, in transgenic mice overexpressing a human τ isoform with four repeated sequences, axonal degeneration develops in the brain and dorsal root ganglia, and is related to accumulation of neurofilaments (Spittaels et al, 1999).…”

Microtubule binding of the Drosophila DMAP‐85 protein is regulated by phosphorylation in vitro

“…A direct interaction of DMAP‐85 with MTs has been previously characterized [32,33]. In this work we test whether DMAP‐85 is phosphorylated and whether binding to MTs is affected by the phosphorylation state of the protein.…”

“…In previous reports we have shown that DMAP-85 stabilizes MTs, promotes MT assembly [32] and associates to stable MTs in late Drosophila embryos [1]. In this report we examine the post-translational modi¢cations of this protein during developmental stages known to be characterized by rapid mitotic divisions.…”

“…To explore further the regulatory activities of MAPs upon MT behavior, we have isolated DMAP-85, a Drosophila 85 kDa MAP present in embryos, larvae and adults, as a protein that can associate to taxol-stabilized MTs [32,33]. The biochemical analyses of DMAP-85 indicated that this protein cosediments with tubulin and promotes tubulin polymerization in vitro through its interaction with exposed domains of MTs, speci¢cally with MAPs binding sequences present at the C-terminal end of L-tubulin [1,32,33]. These results suggest that one of the functions of DMAP-85 could be to regulate the dynamics of MT assembly in the cell.…”

Human MAP tau based targeted cytolytic fusion proteins

“…Tau proteins are found in many animal species, including Drosophila [25], rodents [26], goat [27], goldfish [28] and humans [29]. Discovered in 1975 by Weingarten et al by co-purification with tubulin [30], MAP tau was recognized as a protein factor essential for microtubule assembly [31].…”