title: Tubulin potentiates the interaction of the metalloendopeptidase nardilysin with the neuronal scaffold protein p42IP4/centaurin-α1 (ADAP1).
abstract: We found colocalization of the neuronal protein p42(IP4) (centaurin-α1; ArfGAP with dual pleckstrin homology domain [ADAP1]), the metalloendopeptidase nardilysin (NRD; involved in axonal maturation and myelination) and tubulin in the cytosol and at the plasma membrane of SH-SY5Y neuroblastoma cells. To examine the importance of tubulin for the interaction of NRD with p42(IP4), we treated cells with nocodazole, which interferes with tubulin polymerization. Nocodazole did not affect the colocalization of p42(IP4) and tubulin but caused a clear redistribution of the proteins in cells, so that the colocalization of p42(IP4), tubulin and NRD was visible exclusively in multiple foci. To reveal the mechanism of the interaction between NRD, p42(IP4) and tubulin observed in neuronal cells, we performed Far-Western blotting, a technique that directly detects protein-protein interactions on Western blots. This technique demonstrated that tubulin enhanced the binding of NRD to functionally renatured p42(IP4). The mutation of a highly conserved cysteine residue in NRD to alanine abolished the potentiation by tubulin. NRD lacking the characteristic acidic domain was able to bind p42(IP4) but addition of tubulin did not significantly potentiate the binding of this deletion mutant to p42(IP4). A function-abolishing mutation of the Zn(2+)-binding motif of NRD did not affect the potentiation by tubulin. Thus, the capacity of tubulin to enhance the interaction between p42(IP4) and NRD together with the known interaction of p42(IP4) with F-actin support the novel notion that p42(IP4) plays a possible role as a linker between the two networks, actin and tubulin, in neural cells.
authors.affiliation: Institut für Neurobiochemie, Medizinische Fakultät der Otto-von-Guericke-Universität Magdeburg, Magdeburg, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Borrmann
authors.forename: Rolf
authors.initials: R
authors.lastname: Stricker
authors.forename: Georg
authors.initials: G
authors.lastname: Reiser
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D057809:HEK293 Cells; D006801:Humans; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D009417:Nerve Tissue / Q000166:cytology / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D014404:Tubulin / Q000235:genetics / Q000378:metabolism*; D050257:Tubulin Modulators / Q000494:pharmacology
chemical_list: C100956:ADAP1 protein, human; D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D009419:Nerve Tissue Proteins; D014404:Tubulin; D050257:Tubulin Modulators; D008666:Metalloendopeptidases; C087976:nardilysin; D015739:Nocodazole