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ACTH-induced caveolin-1 tyrosine phosphorylation is related to podosome assembly in Y1 adrenal cells

Departamento de Bioquímica Humana, Facultad de Medicina, Universidad de Buenos Aires, C1121ABG Buenos Aires, Argentina.
Experimental Cell Research (Impact Factor: 3.25). 05/2005; 304(2):432-42. DOI: 10.1016/j.yexcr.2004.11.019
Source: PubMed

ABSTRACT

Y1 adrenocortical cells respond to ACTH with a characteristic rounding-up that facilitates cAMP signaling, critical for transport of cholesterol to the mitochondria and increase in steroid secretion. We here demonstrate that caveolin-1 participates in coupling activation of protein kinase A (PKA) to the control of cell shape. ACTH/8-Br-cAMP induced reorganization of caveolin-1-positive structures in correlation with the cellular rounding-up. Concomitant with this change, there was an increase in the phosphorylation of caveolin-1 (Tyr-14) localized at focal adhesions (FA) with reorganization of FA to rounded, ringlike structures. Colocalization with phalloidin showed that phosphocaveolin is present at the edge of actin filaments and that after ACTH stimulation F-actin dots at the cell periphery become surrounded by phosphocaveolin-1. These observations along with electron microscopy studies revealed these structures as podosomes. Podosome assembly was dependent on both PKA and tyrosine kinase activities because their formation was impaired after treatment with specific inhibitors [myristoylated PKI (mPKI) or PP2, respectively] previous to ACTH/8-Br-cAMP stimulation. These results show for the first time that ACTH induces caveolin-1 phosphorylation and podosome assembly in Y1 cells and support the view that the morphological and functional responses to PKA activation in steroidogenic cells are related to cytoskeleton dynamics.

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    • "We found that the ring of podosomes in microglia was enriched in p-Tyr14Cav1. Cav1 has only been reported in invadopodia in three cell lines; that is, the nonphosphorylated form in two cancer cell lines [78,79], and the phosphorylated form in an ACTH-stimulated adrenal cell line [33]. Nox1 generates reactive oxygen species, which can activate protein kinases and inhibit protein tyrosine phosphatases [80]. "
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    • "Then, cells were fixed with 4% paraformaldehyde in PBS for 10 minutes at room temperature and permeabilized with blocking solution (0.3% Triton X-100 and 1% BSA in PBS) for 60 minutes at room temperature. The detailed procedure was described previously [59]. Cells were incubated with anti-pERK1/2 cy2-conjugated antibody (1∶250) overnight at 4°C. "
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