REDISTRIBUTION AND ENHANCED PROTEIN-KINASE C-MEDIATED PHOSPHORYLATIONOF ALPHA-ADDUCIN AND GAMMA-ADDUCIN DURING RENAL TUMOR PROGRESSION

Tumor promotion/progression is known to be due in part to increased si gnaling through a variety of mitogenic pathways, including protein kin ase C (PKC), To determine whether increased PKC activity could play a role in promotion and progression of renal cancer, we monitored PKC ac tivity in normal and progressively transformed renal neoplasias from E ker rats. Eker rats carry a defect in the tumor suppressor TSC2 gene t hat predisposes them to renal carcinoma, whereas additional factors in fluence tumor promotion/progression in accordance with a 'two-hit'' mo del. We used the phosphorylation of adducins at Ser-660, a known PKC p hosphorylation site, as a reporter for endogenous PKC activity. In nor mal proximal tubules, total adducin levels (measured with a phosphoryl ation state-insensitive antibody) were relatively high, whereas pSer66 0-adducin (measured with a phosphorylation state-sensitive antibody) l evels were very low. In comparison, in renal carcinomas, total adducin levels were decreased, and pSer-660-adducin levels were increased. Ch anges in phosphorylation correlated with changes in localization. In n ormal tissue, alpha- and gamma-adducin are targeted to the apical and basal membranes of proximal tubules, respectively, implying unique fun ctions for these related proteins. In early lesions (atypical tubules) , differential targeting is lost, and both alpha- and gamma-adducins l ocalize to the basal membrane. In more advanced lesions, staining in l ateral membranes at cell-cell contacts becomes apparent. Furthermore, in cells that have lost basement membrane contact, plasma membrane tar geting is no longer apparent. These changes in adducin expression leve ls, phosphorylation state, and localization parallel the increased gro wth potential and dedifferentiation of the progressive tumor phenotype s. These data demonstrate the utility of phosphorylation state-selecti ve antibodies in immunohistochemical applications as reporters of endo genous PKC activity in tissue samples. We also provide the first evide nce that increased PKC activity and phosphorylation of important targe t proteins occurs during progressive transformation in a non-phorbol e ster tumor promotion model in vivo.