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Abstract: SA-PO075

Regulation of Epidermal Growth Factor Induced Rac1 Activity by the Adapter Protein Dok-4

Session Information

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Lemay, Serge, Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada
  • Roodman, Victoria, Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada
  • Baldwin, Cindy, Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada
  • Masztalerz, Agnieszka, Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada
  • Namkung, Yoon, Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada
  • Laporte, Stephane A., Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada
  • Takano, Tomoko, Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada
Background

Following renal ischemia-reperfusion injury (IRI), locally produced or exogenously administered growth factors such as EGF promote tubular repair. To better understand the intracellular signaling events that may regulate this response, we explored the role of Dok-4, a highly conserved member of the Dok family of adapter proteins most abundantly expressed in epithelial cells and thought to mediate inhibitory signaling through largely undefined molecular interactions.

Methods

We screened a kidney cDNA library by yeast two-hybrid using Dok-4 as bait, with co-expression of the tyrosine kinase Lyn in order to detect phspho-Tyr (pY)-dependent interactions. Interactions were validated and their structural basis mapped by mutagenesis, co-IP and pull-down in transfected 293 cells. Expression of relevant proteins was confirmed in mouse kidneys subjected to 30 min. of unilateral ischemia and in sham controls. A novel bioluminescence resonance energy transfer (BRET)-based Rac1 assay was used to study the impact of Dok-4/Chn2 interaction in on EGF-induced Rac1 activity.

Results

The Rac1 GTPase activating protein (GAP) β2-chimerin (Chn2) was identified as a Dok-4 partner. This interaction involved binding of the Dok-4 PTB domain to phosphorylated Y153 of Chn2. Notably, Chn2 Y153 is contained within a canonical PTB-binding motif (NPXY) that is conserved in the highly homologous Rac-GAP α2-chimerin (Chn1), where pY143 can also mediate binding to Dok-4. While Dok-4 protein is expressed in kidney tissue and all cultured tubular cells examined to date, Chn2 protein was undetectable except in cerebellum. However, 24h after renal IRI, Chn2 was strongly expressed. BRET-based quantification of GTPase activity in 293 cells showed a rapid activation of Rac1 by EGF. Chn2 attenuated this activation in the presence of Dok-4 WT, but not mutant Dok-4 lacking the membrane-targeting PH domain. Retargeting of mutant Dok-4 to the membrane by a myristoylation signal rescued the cooperative inhibition of Rac1 by Dok-4 and Chn2.

Conclusion

The Rac-GAP Chn2 is a novel pY-dependent Dok-4 partner and effector expressed in the injured kidney. Dok-4 facilitates inhibition of Rac1 by promoting recruitment of Chn2 at the membrane. This cooperative inhibition of Rac1 may regulate key downstream events in renal IRI, including proliferation, migration and tubular repair.

Funding

  • Private Foundation Support