Abstract: TH-PO355

Proximity-Ligation Assay Identified the Rho Guanine Nucleotide Exchange Factor, β-PIX, as a Rac1-Interactor in Podocytes

Session Information

Category: Cell Biology

  • 201 Cell Signaling, Oxidative Stress

Authors

  • Maier, Mirela, McGill University , Montreal, Quebec, Canada
  • Aoudjit, Lamine, McGill University , Montreal, Quebec, Canada
  • Baldwin, Cindy, McGill university, Montreal, Alberta, Canada
  • Takano, Tomoko, None, Montreal, Quebec, Canada
Background

Hyperactivity of Rac1 (a small GTPase) in podocytes has been implicated in the development of proteinuria and focal segmental glomerulosclerosis (FSGS). We sought to identify guanine nucleotide exchange factors (GEFs) that activate Rac1 in podocytes.

Methods

BioID, a proximity-based ligation assay, was used to identify Rac1-GEFs in human podocytes (HP). This assay consists of HP expressing a bait, Rac1G15A (a mutant of Rac1 reported to have high affinity to active GEFs), conjugated to a biotin ligase, BirA (BirA-Rac1G15A). BirA alone was used as control. HP were incubated with biotin for 18 hours in the culture medium, and biotinylated proteins (i.e. proteins that have come in close proximity of Rac1G15A) were isolated with streptavidin-beads and identified by mass spectroscopy. Active Rac1 was visualized with immunofluorescence staining (IF) and quantified using CRIB pulldown (PD). β-PIX binding to Rac1 was determined by PD with GST-Rac1, followed by immunoblotting (IB).

Results

BioID identified 5 GEFs in BirA-Rac1G15A expressing HP; β-PIX was by far the most abundant and the only one whose quantity was consistently enriched (20-fold or more) in three independent experiments, as compared with control cells expressing BirA alone. Thus, we proceeded to characterize β-PIX in podocytes. By IB, HP expressed only one isoform of β-PIX (75 kDa), whereas mouse podocytes (MP) expressed an additional isoform of 88kDa. These isoforms were verified by RT-PCR. By IF of kidney sections, β-PIX was expressed in human, mouse, and rat glomeruli, largely overlapping with nephrin and/or β1-integrin. By IF of un-stimulated MP, β-PIX was localized diffusely in the cytosol and at cell periphery, which, upon stimulation with epidermal growth factor (EGF), shifted to the tip of cellular projections, co-localizing with active Rac1. Biochemically, EGF increased Rac1 activity, and β-PIX binding to Rac1 by 5 and 10 minutes (5min: 1.56-fold ±0.24; 10min: 1.34-fold ±0.13, n=5, p<0.05).

Conclusion

BioID using RacG15A as bait identified β-PIX as a predominant Rac1-interactor in HP. β-PIX is expressed in podocytes, and its binding to Rac1 is modulated by EGF. Together, this data warrants further studies on the functional role of β-PIX in podocyte health and disease.

Funding

  • Government Support - Non-U.S.