Transformation-sensitive changes in expression, localization, and phosphorylation of adducins in renal proximal tubule epithelial cells.
Adducins are cytoskeletal proteins that facilitate interactions between spectrin and actin to form the subcortical membrane skeleton. We recently determined that alpha- and gamma-adducins are among a group of PKC substrates that we have designated "STICKS" (substrates that interact with C-kinase). To study the role of adducins and their regulation by protein kinase C (PKC) in carcinogenesis, we compared the content, localization, and phosphorylation of alpha- and gamma-adducins in primary renal proximal tubule epithelial (RPTE) cells and oncogene-altered derivative lines. RPTE cells expressing adenovirus E1A are immortalized but not transformed, whereas RPTE cells expressing SV40 large T antigen are transformed. Phosphorylation of adducins was monitored with a phosphorylation state-specific antibody directed toward the PKC phosphorylation site on adducins. Basal levels of phospho-alpha-adducin were relatively low in growing and confluent primary RPTE cells; however, basal levels of phosphoadducins relative to total adducins were increased in E1A-RPTE and SV40-RPTE cells. Phorbol esters stimulated alpha-adducin phosphorylation to a greater extent in primary cells than in oncogene-altered cells, possibly because of the already high basal levels of phosphorylation in those cells. Phosphorylated adducins were preferentially recovered in the soluble fraction, indicating that PKC phosphorylation either directly or indirectly influences the subcellular location and functions of adducins in regulating membrane skeleton structure. Thus, these studies provide evidence for increased endogenous PKC activity in oncogene-altered cells and suggest that the increased activity directly influences cytoskeletal organization by phosphorylating regulatory proteins, such as the adducins.
Fowler
L
L
Adirondack Biomedical Research Institute, Lake Placid, New York 12946, USA.
Dong
L
L
Bowes
R C
RC
van de Water
B
B
Stevens
J L
JL
Jaken
S
S
D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000737:chemistry; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D003593:Cytoplasm / Q000737:chemistry; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000378:metabolism*; D008297:Male; D009857:Oncogenes; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D000099291:Adducins
D002148:Calmodulin-Binding Proteins; D011493:Protein Kinase C; D000099291:Adducins