3 research outputs found
Angiotensin II regulates phosphorylation of actin-associated proteins in human podocytes
Within the kidney, angiotensin II (AngII) targets different cell types in the vasculature, tubuli, and glomeruli. An important part of the renal filtration barrier is composed of podocytes with their actin-rich foot processes. In this study, we used stable isotope labeling with amino acids in cell culture coupled to mass spectrometry to characterize relative changes in the phosphoproteome of human podocytes in response to short-term treatment with AngII. In 4 replicates, we identified a total of 17,956 peptides that were traceable to 2081 distinct proteins. Bioinformatic analyses revealed that among the increasingly phosphorylated peptides are predominantly peptides that are related to actin filaments, cytoskeleton, lamellipodia, mammalian target of rapamycin, and MAPK signaling. Among others, this screening approach highlighted the increased phosphorylation of actin-bundling protein, L-plastin (LCP1). AngII-dependent phosphorylation of LCP1 in cultured podocytes was mediated by the kinases ERK, p90 ribosomal S6 kinase, PKA, or PKC. LCP1 phosphorylation increased filopodia formation. In
addition, treatmentwith AngII led to LCP1 redistribution to the cell margins,membrane ruffling, and formation of lamellipodia. Our data highlight the importance of AngII-triggered actin cytoskeleton-associated signal transduction in podocytes
LIM and SH3 protein 1 (LASPâ1): A novel link between the slit membrane and actin cytoskeleton dynamics in podocytes
The foot processes of podocytes exhibit a dynamic actin cytoskeleton, which maintains their complex cell structure and antagonizes the elastic forces of the glomerular capillary. Interdigitating secondary foot processes form a highly selective filter for proteins in the kidney, the slit membrane. Knockdown of slit membrane components such as Nephrin or Neph1 and cytoskeletal adaptor proteins such as CD2AP in mice leads to breakdown of the filtration barrier with foot process effacement, proteinuria, and early death of the mice. Less is known about the crosstalk between the slit membraneâassociated proteins and cytoskeletal components inside the podocyte foot processes. Our study shows that LASPâ1, an actinâbinding protein, is highly expressed in podocytes. Electron microscopy studies demonstrate that LASPâ1 is found at the slit membrane suggesting a role in anchoring slit membrane components to the actin cytoskeleton. Live cell imaging experiments with transfected podocytes reveal that LASPâ1 is either part of a highly dynamic granular complex or a static, actin cytoskeletonâbound protein. We identify CD2AP as a novel LASPâ1 binding partner that regulates its association with the actin cytoskeleton. Activation of the reninâangiotensinâaldosterone system, which is crucial for podocyte function, leads to phosphorylation and altered localization of LASPâ1. In vivo studies using the Drosophila nephrocyte model indicate that Lasp is necessary for the slit membrane integrity and functional filtration