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.

In vitro reconstitution reveals phosphoinositides as cargo-release factors and activators of the ARF6 GAP ADAP1

“…We found that ADAP1 had a weak but detectable affinity toward microtubules (Fig. 2 A, i), consistent with an earlier report (5). The presence of AMP-PNP-bound, unlabeled KIF13B strongly increased the fluorescence signal of mWasabi-ADAP1 (Fig.…”

“…For example, in the developing neuron, the protein ADAP1 (ADP-ribosylation factor GTPase-activating protein [ArfGAP] with dual pleckstrin homology [PH] domains 1) has been suggested to control dendrite branching by regulating the small GTPase ARF6. Together with the motor protein KIF13B, ADAP1 is also thought to mediate delivery of the second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ) to the axon tip, thus contributing to PIP 3 polarity. However, what defines the function of ADAP1 and how its different roles are coordinated are still not clear.…”

“…microtubule transport | PIP 3 signaling | small GTPases | in vitro reconstitution | neuronal development N eurons are highly polarized cells with one axon and several highly branched dendrites (1). For their development, the protein ADAP1 (ADP-ribosylation factor GTPase-activating protein [ArfGAP] with dual pleckstrin homology [PH] domains 1; synonym, centaurin-α1) has been found to play an important role as it is thought to connect the major regulatory players involved (2): First, it contains two PH domains that can bind the phosphoinositide phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ) present in the plasma membrane and in the case of the C-terminal PH domain also to PI(3,4)P 2 (Fig. 1A) (3).…”

“…Second, it has a putative ARF GAP domain at its N terminus that is thought to regulate the activity of the small GTPase ARF6 (2,4). Third, it was found to interact with the microtubule cytoskeleton and the kinesin-3 member KIF13B (3,5,6). How these various interactions are coordinated and contribute to the cellular architecture during neuronal development is not well-understood.…”

A20

“…In the brain, ADAP1 was found in neurons of the cortex, hypothalamus, and hippocampus (Sedehizade et al 2002;Moore et al 2007). SH-SY5Y neuronal cell line has been used to study the effects of ADAP1 expression and localization in vitro (Borrmann et al 2011a;Borrmann et al 2011b;Szatmari et al 2013).…”