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Abstract: SA-PO619

Ouabain Regulated Phosphoproteome Reveals Molecular Mechanisms Behind Na,K-ATPase Control of Cell Adhesion, Proliferation, and Survival of Renal Cells

Session Information

  • Pharmacology
    October 27, 2018 | Location: Exhibit Hall, San Diego Convention Center
    Abstract Time: 10:00 AM - 12:00 PM

Category: Pharmacology (PharmacoKinetics, -Dynamics, -Genomics)

  • 1700 Pharmacology (PharmacoKinetics, -Dynamics, -Genomics)

Authors

  • Scott, Lena, Karolinska Institutet, Stockholm, Sweden
  • Zhang, Liang, Karolinska Institutet, Stockholm, Sweden
  • Fontana, Jacopo Maria, Karolinska Institutet, Stockholm, Sweden
  • Svensson, Daniel, Karolinska Institutet, Stockholm, Sweden
  • Brismar, Hjalmar, Royal Institute of Technology, Stockholm, Sweden
  • Aperia, Anita, Karolinska Institutet, Stockholm, Sweden
Background

Ouabain, a Na,K-ATPase ligand, triggers calcium release and Erk phosphorylation. Ouabain protects from apoptosis and stimulates proliferation and cell contact in primary and immortalized kidney epithelial cells. However, the mechanisms that connect the calcium and Erk signaling with the downstream effects are unknown.

Methods

We have performed a proteomic and phosphoproteomic study on COS cells, derived from embryonic monkey kidney. Cells were treated with ouabain for 10 and 20 min in concentrations that do not increase [Na+]i. Hierarchical clustering was applied to describe the temporal pattern of phosphorylation events. Gene ontology analysis was then used to examine whether phosphorylation events with different temporal patterns regulate distinct cellular processes. To verify the significance of identified proteins, siRNA studies were performed on primary rat proximal tubule cells.

Results

We identified 1941 ouabain-regulated sites that were either phosphorylated or dephosphorylated in a transient or sustained manner.
The state of phosphorylation for proteins associated with cell adhesion and proliferation was prominently altered by ouabain. Analysis of protein-protein interaction networks showed a high connectivity of phosphoproteins that regulate cell adhesion and cell proliferation.
Protein kinases and calcium dependent protein kinases were enriched among the proteins where the phosphorylation state is regulated by ouabain. Downregulation of protein expression by siRNA showed that CaMKK1 and CaMK2γ are essential for ouabain protection from apoptosis caused by serum deprivation or exposure to high glucose

Conclusion

This study represents a major advancement in the understanding of the dual function of Na,K-ATPase as an ion pump and as a signal transducer. It offers an insight into the role of ouabain for kidney growth and development and for protection against apoptosis.

Funding

  • Private Foundation Support