PKC-z phosphorylates the Na+,K+-ATPase a-subunit and associates with clathrin vesicles during subunit endocytosis in response to dopamine

MENDEZ, C.F.; LEIBIGER, B.; EFENDIEV, R.; PEDEMONTE, C.; KATZ, A.; LEIBIGER, I.; BERTORELLO, A.M.

Toronto

Congreso; 33rd Annual meeting of the American Society of Nephrology; 2000

The American Society of Nephrology

Inhibition of Na+,K+-ATPase (NK) activity by G proteín-coupled receptor signals in renal epithelial cells is mediated by endocytosis of active enzyme molecules via a protein kinase C-dependent mechanism. We sought here to identify the PKC isoform(s) involved in this process, and to determine whether activation of such PKC isoform(s) results in phosphorylation of NK molecules. Incubation of rat proximal tubule cells or an opossum kidney (OK) cell line with 1 mM dopamine (D) increased PKC-z activity determined as the degree of autophosphorylation of its T410 residue. NK activity, determined as ouabain-sensitive 86Rb+ transport, was significantly inhibited by D, and this effect was blocked when cells were incubated in the presence of a PKC-z inhibitor peptide. Phosphorylation of the NK a-subunit was significantly increased by D, and this effect was also abolished by coincubation with the PKC-z inhibitor peptide. The subcellular localization of PKC-z was studied by confocal laser scanning microscopy of an engineered fusion between PKC-z and green fluorescent protein (GFP). On-line monitoring of GFP-tagged PKC-z in living cells incubated with D showed a rapid (within 1 min) change in its distribution pattern in selected areas of the cell. Confocal microscopy analysis of fixed OK cells treated with D revealed colocalization of PKC-z with clathrin; this increase of PKC-z in clathrin vesicles was also seen by Western blot analysis of purified vesicles. <!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:595.3pt 841.9pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:35.4pt; mso-footer-margin:35.4pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> In conclusion, these data indicate that in renal epithelial cells derived from proximal tubules PKC-z phosphorylates the NK a-subunit and promotes its endocytosis, and that the complex PKCz-NK internalizes via a clathrin vesicle-dependent mechanism.