Platelet-derived growth factor and angiotensin II induce different spatial distribution of protein kinase C-alpha and -beta in vascular smooth muscle cells.

Abstract

Protein kinase C is an important second-messenger system that is translocated from the cytosol to the cell membrane on cell stimulation. We used confocal microscopy to study the spatial distribution of protein kinase C isoforms after stimulation of cultured vascular smooth muscle cells with platelet-derived growth factor and angiotensin II (Ang II). Monoclonal antibodies for the isoforms a and p were used. Translocation was also assessed by Western blot. Isoform a was evenly distributed in the cytosol, whereas the £ isoform formed coarse granules in the perinuclear region. Both isoforms shifted from the cytosolic to the membrane fraction after exposure to Ang II (10~7 mol/L) and platelet-derived growth factor (100 ng/mL at 6, 12, and 20 minutes). Confocal microscopy showed a rapid assembly of isoform a along P rotein kinase C (PKC) is a group of calcium-and phospholipid-dependent protein kinases (isoforms) involved in signal transduction responses.1 Activation of PKC has been implicated in vascular smooth muscle cell (VSMC) contraction, 2 DNA synthesis, and cell growth 36 (for review see Reference 7). PKC is located in the cytosol of VSMCs and other cells during the resting state but is "translocated" to the cell membrane after stimulation. 810 However, because the membrane fraction includes not only cell membranes but also nuclear material, earlier studies do not allow a precise definition of subcellular PKC distribution after cell stimulation. Other investigators observed that only a portion of PKC is shifted to the surface membranes and that PKC is associated with nuclear membranes and cytoskeletal proteins, 11 1 5 which raises the possibility that PKC may also perform important tasks elsewhere within the cell.The pattern of isoform distribution may also vary depending on the activation state of the cell or the agonists used. 16 In VSMCs the calcium-sensitive PKC isoforms a and )3 9 as well as the calcium-independent PKC isoforms e and £ have been described. 17 We tested the hypothesis that PKC isoforms have different distributions in VSMCs at rest and during agonist stimulation. We used confocal microscopy to examine the From the UniversitatskJinikum Steglitz and UniversitStsklinikum Rudolf Virchow, Franz Volhard Klinik at the Max-Delbruck Center for Molecular Medicine, Free University of Berlin (Germany).Correspondence to Hermann Haller, MD, Franz Volhard Klinik, Universitatsklinikum Rudolf Virchow, Wiltberg Strasse 50, 13125 Berlin, FRG. cytosolic fibers at 6 minutes followed by a translocation toward the nucleus at 12 minutes with Ang II. Platelet-derived growth factor engendered a similar response; however, a cytoskeletal distribution was not observed. The /3 isoform was rapidly translocated by both inducers to the perinuclear region and the nucleus. Our results show that inducers cause a translocation of protein kinase C isoforms not only into the cell membrane but also into the cell nucleus. We suggest that protein kinase C may also be important for nuclear signaling. (Ang II) and platelet-d...

The Proliferative Effect of Vascular Endothelial Growth Factor Requires Protein Kinase C-α and Protein Kinase C-ζ

“…This observation could indicate that VEGF induces a translocation of PKC-␣ into the nucleus. Nuclear translocation of this isoform by tyrosine kinase receptor signaling has been reported previously by us 21,30,31 and others. [32][33][34] Which signal directs PKC isoforms to the nucleus is presently unclear.…”

Aldosterone Potentiates Angiotensin II–Induced Signaling in Vascular Smooth Muscle Cells

“…12 Passages 2 to 4 were used for immunohistochemistry and passage 4 to 10 for Western blotting. VSMCs were phenotyped by staining for muscle-specific ␣-actin (Dako) and desmin (Boehringer-Mannheim).…”

“…12 At least 50 to 80 cells from each experiment were examined under each condition by 2 different investigators without knowledge of the origin of the specimens. Quantification was performed with histogram function in the MRC Laser Sharp software.…”

Angiotensin AT 1 Receptor Stimulates Heat Shock Protein 27 Phosphorylation In Vitro and In Vivo

“…Because Ang II activates protein kinase C (PKC) in VSMCs 17 and direct activation of PKC has been shown to increase the phosphorylation of small heat shock proteins, 18 the role of PKC in Ang II-stimulated phosphorylation was examined. Cells were pretreated with the PKC inhibitor GF 109203X (GFX, 5 mol/L) for 15 minutes followed by stimulation with either Ang II or phorbol 12-myristate 13-acetate (PMA) for 15 minutes.…”