Regulation of P- ATPases through interaction with acetylated tubulin.

CHESTA M. E.; ZAMPAR G. G.; CARBAJAL A.; CASALE C. H.; ARCE C. A

Santa Cruz

Workshop; Emerging Concepts on Neuronal Cytoskeleton; 2011

Instituto de Dinámica Celular y Biotecnología (ICDB)

P-ATPases are a group of membrane pumps that translocate different ions against their electrochemical gradients using the energy provided by ATP hydrolysis. Several of these P-ATPases (including the Na+/K+-ATPase, the plasma membrane Ca2+ ATPase [PMCA] and the H+-ATPase) were shown to interact with acetylated tubulin resulting in inhibition of their enzymatic activities. Now, we show a molecular characterization of the interaction with the Na+/K+-ATPase. Our results indicate that the association is mediated by the fifth cytoplasmic domain (CD5) of the alpha subunit of Na+/K+-ATPase. This conclusion is supported by four observations: 1) a 17-amino acid peptide corresponding to the sequence of CD5 associates with  tubulin in pull-down assays; 2) retained tubulin is mainly (if not exclusively) of the acetylated isotype; 3) the interaction is inhibited by solubilized membranes that contain full-length Na+/K+-ATPase; and 4) after covalent cross-linking of CD5 with a tubulin preparation and subsequent SDS-PAGE, the CD5 migrated in a single band located slightly above the acetylated tubulin band. Taken together, these evidences strongly support the idea that CD5 mediates the interaction between Na+/K+-ATPase and acetylated tubulin in a direct manner. Preliminary results also suggest that the CD2 and CD3 of PMCA may also be involved in the direct association with acetylated tubulin. These interactions may have important implications on organism physiology. In this regard, we found that human erythrocytes from hypertensive subjects present a partially inhibited Na+/K+-ATPase activity and an increased content of the ATPase/acetylated tubulin complex as compared with erythrocytes from normal subjects.