Endothelial cell tyrosine kinase receptor and G protein-coupled receptor activation involves distinct protein kinase C isoforms.
Protein kinase C (PKC) is a family of serine/threonine protein kinase isoforms that is important to intracellular enzymes for both tyrosine kinase receptors and G protein coupled receptors. However, which isoforms are linked to which class of receptors in endothelial cell signaling is not known. Moreover, the PKC isoforms in endothelial cells have not been thoroughly characterized. We tested the hypothesis that specific PKC isoforms are involved in different signaling pathways. PKC isoform expression was assessed by using reverse transcription polymerase chain reaction and Western blotting. The spatial distribution of PKC after stimulation of the cells with basic fibroblast growth factor (bFGF) and thrombin was examined by using confocal microscopy. Expression of PKC alpha, delta, epsilon, theta, and zeta was detectable on both the mRNA and protein levels. In resting cells, PKC alpha and epsilon were mostly distributed in the cytosol, while PKC alpha and epsilon were also present in the nucleus. Nuclear immunoreactivity of PKC alpha and epsilon increased significantly between passages 1 and 3. The phorbol ester TPA induced a rearrangement of PKC delta and a translocation of PKC alpha and epsilon to the nucleus. Treatment of endothelial cells with TPA for 24 hours caused PKC alpha, delta, and epsilon to disappear, while PKC zeta was not influenced by TPA. bFGF induced a rapid assembly of PKC alpha along cytosolic structures, followed by a translocation of the isoform toward the perinuclear region and into the nucleus. bFGF had a smaller effect on PKC epsilon. In contrast, thrombin had a similar effect on nuclear translocation of PKC alpha, did not influence PKC epsilon, and induced a rapid nuclear translocation of PKC zeta. Thus, tyrosine kinase receptor activation via bFGF induced a rapid association of PKC alpha and epsilon with nuclear structures, while activation of the G protein-coupled thrombin receptor increased mostly nuclear PKC zeta. The translocation of PKC isoforms into the nucleus by growth-promoting factors may be important for the induction of endothelial cell growth.
Haller
H
H
Franz Volhard Clinic, Berlin, Germany.
Ziegler
W
W
Lindschau
C
C
Luft
F C
FC
D001483:Base Sequence; D015153:Blotting, Western; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000378:metabolism*; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011493:Protein Kinase C / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000187:drug effects / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology; D014158:Transcription, Genetic
D007527:Isoenzymes; D015335:Molecular Probes; D016222:Fibroblast Growth Factor 2; D020794:Receptor Protein-Tyrosine Kinases; D011493:Protein Kinase C; D013917:Thrombin; D019204:GTP-Binding Proteins