PKC-alpha-mediated remodeling of the actin cytoskeleton is involved in constitutive albumin uptake by proximal tubule cells.
One key role of the renal proximal tubule is the reabsorption of proteins from the glomerular filtrate by constitutive receptor-mediated endocytosis. In the opossum kidney (OK) renal proximal tubule cell line, inhibition of protein kinase C (PKC) reduces albumin uptake, although the isoforms involved and mechanisms by which this occurs have not been identified. We used pharmacological and molecular approaches to investigate the role of PKC-alpha in albumin endocytosis. We found that albumin uptake in OK cells was inhibited by the pan-PKC blocker bisindolylmaleimide-1 and the isoform-specific PKC blockers Go-6976 and 2',3,3',4,4'-hexahydroxy-1,1'-biphenyl-6,6'-dimethanol dimethyl ether, indicating a role for PKC-alpha. Overexpression of a kinase deficient PKC-alpha(K368R) but not wild-type PKC-alpha significantly reduced albumin endocytosis. Western blot analysis of fractionated cells showed an increased association of PKC-alpha-green fluorescent protein with the membrane fraction within 10-20 min of exposure to albumin. We used phalloidin to demonstrate that albumin induces the formation of clusters of actin at the apical surface of OK cells and that these clusters correspond to the location of albumin uptake. These clusters were not present in cells grown in the absence of albumin. In cells treated either with PKC inhibitors or overexpressing kinase-deficient PKC-alpha(K368R) this actin cluster formation was significantly reduced. This study identifies a role for PKC-alpha in constitutive albumin uptake in OK cells by mediating assembly of actin microfilaments at the apical membrane.
Hryciw
Deanne H
DH
School of Biomedical Sciences, Univ. of Queensland, St Lucia, 4072, Queensland, Australia.
Pollock
Carol A
CA
Poronnik
Philip
P
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D000418:Albumins / Q000493:pharmacokinetics*; D000818:Animals; D002227:Carbazoles / Q000494:pharmacology; D004705:Endocytosis / Q000502:physiology; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D008301:Maleimides / Q000494:pharmacology; D009893:Opossums; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D014162:Transfection
D000199:Actins; D000418:Albumins; D002227:Carbazoles; D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; C081021:Go 6976; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; C070515:bisindolylmaleimide I