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Tubulin potentiates the interaction of the metalloendopeptidase nardilysin with the neuronal scaffold protein p42IP4/centaurin-α1 (ADAP1)

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Abstract

We found colocalization of the neuronal protein p42IP4 (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 p42IP4, we treated cells with nocodazole, which interferes with tubulin polymerization. Nocodazole did not affect the colocalization of p42IP4 and tubulin but caused a clear redistribution of the proteins in cells, so that the colocalization of p42IP4, tubulin and NRD was visible exclusively in multiple foci. To reveal the mechanism of the interaction between NRD, p42IP4 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 p42IP4. 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 p42IP4 but addition of tubulin did not significantly potentiate the binding of this deletion mutant to p42IP4. A function-abolishing mutation of the Zn2+-binding motif of NRD did not affect the potentiation by tubulin. Thus, the capacity of tubulin to enhance the interaction between p42IP4 and NRD together with the known interaction of p42IP4 with F-actin support the novel notion that p42IP4 plays a possible role as a linker between the two networks, actin and tubulin, in neural cells.

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Abbreviations

AD:

Alzheimer’s disease

ADAM:

A disintegrin and metalloendopeptidase

Aβ:

Amyloid β-peptide

ADAP:

ArfGAP (GTPase activating protein) with dual pleckstrin homology domain

APP:

Amyloid precursor protein

BACE:

β-Secretase

CNP:

2',3'-Cyclic-nucleotide 3'-phosphodiesterase

DAC:

Acidic domain

DMEM:

Dulbecco’s modified Eagle’s medium

FCS:

Fetal calf serum

FSBB:

FCS-containing blocking buffer

GFP:

Green fluorescent protein

HEK293:

Human embryonic kidney 293 cells

NRD1:

Nardilysin

PBS:

Phosphate-buffered saline

PFA:

Paraformaldehyde solution

RanBPM:

Ran-binding protein M

RA:

All-trans retinoic acid

Sf9:

Spodoptera frugiperda

TACE:

TNF-α-converting enzyme

TNF:

Tumor necrosis factor

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Acknowledgements

We are grateful to M. Aswendt for help with the cloning of hsNRD1, to Dr. A. Schneider for help with confocal microscopy, to Dr. T. Hanck for the plasmid pEGFP-p42-C1 and to Dr. S. Aleshin for helpful discussions.

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Authors and Affiliations

  1. Institut für Neurobiochemie, Medizinische Fakultät der Otto-von-Guericke-Universität Magdeburg, Leipziger Strasse 44, 39120, Magdeburg, Germany

    Claudia Borrmann, Rolf Stricker & Georg Reiser

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  1. Claudia Borrmann
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  2. Rolf Stricker
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  3. Georg Reiser
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Correspondence to Georg Reiser.

Additional information

The work was supported by the German federal state of Sachsen-Anhalt within the Europäischer Fond für regionale Entwicklung (EFRE 2007–2013).

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Fig. S1

Influence of treatment with nocodazole on the morphology of SH-SY5Y cells and localization of NRD (red), p42IP4 (green) and tubulin (blue) within these cells. SH-SY5Y cells were stably transfected with p42IP4-GFP, seeded on coverslips and treated with 10 μM retinoic acid (RA) for 6 days. a Cells were fixed and permeabilized. b Cells were treated with 5 μM nocodazole for 1 h at 37°C before they were fixed and permeabilized for immuncytochemistry. Cells were double-stained with NRD and β-tubulin antibodies and with Alexa Fluor 555, followed by Alexa Fluor 633 antiserum (arrangement of individual images in a as in b). (PDF 136 kb)

Fig. S2

Localization of NRD, p42IP4 and tubulin in SH-SY5Y cells treated with nocodazole. This supplementary figure corresponds to Fig. 2a in the main text. It gives the single confocal images of p42IP4-GFP (green, top left), β-tubulin (blue, bottom left) and NRD (red, top right) in order to complement the merged image. The resulting merged image (bottom right) is identical to Fig. 2a. Conditions for analysis and immuncytochemistry of SH-SY5Y cells stably transfected with p42IP4-GFP are described in the legend of Fig. 2. (PDF 272 kb)

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Borrmann, C., Stricker, R. & Reiser, G. Tubulin potentiates the interaction of the metalloendopeptidase nardilysin with the neuronal scaffold protein p42IP4/centaurin-α1 (ADAP1). Cell Tissue Res 346, 89–98 (2011). https://doi.org/10.1007/s00441-011-1245-z

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