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.