Downregulation of epidermal growth factor receptor signaling by singlet oxygen through activation of caspase-3 and protein phosphatases.
Downregulation of survival signaling pathways contributes to the cytotoxicity of reactive oxygen species (ROS) and may underlie certain therapies for hyperproliferative diseases. We have investigated the role of singlet oxygen, an ROS formed by photosensitization, in the regulation of survival signaling via the epidermal growth factor receptor (EGFR). Exposure of human keratinocytes to singlet oxygen resulted in rapid loss of EGFR, which was not blocked by either inhibition of receptor internalization or by interrupting the major proteolytic pathways (proteasome, lysosome or calpain). However, pretreatment with a caspase-3 inhibitor, DEVD-FMK, inhibited EGFR degradation. Caspase-3 cleavage was detected as early as 5 min after singlet oxygen treatment, and recombinant active caspase-3 completely cleaved EGFR in a keratinocyte membrane fraction. The singlet oxygen-induced loss of EGFR was accompanied by dephosphorylation of EGFR as well as of Akt and extracellular signal-regulated kinase 1/2 (ERK)1/2. Singlet oxygen-induced protein dephosphorylation was not dependent on activation of caspase-3. In contrast, inhibition of protein phosphatases (PPs) with okadaic acid completely blocked dephosphorylation of EGFR, ERK1/2 and Akt as well as degradation of EGFR. These results indicate that the oxidative stress produced by singlet oxygen rapidly disrupts EGFR-mediated signaling by decreasing both the protein level and its phosphorylation. These responses depended on intertwined activation of caspase-3 and PPs.
Zhuang
Shougang
S
Wellman Laboratories of Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
Ouedraogo
Gladys D
GD
Kochevar
Irene E
IE
D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000502:physiology*; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000502:physiology; D019281:Dimerization; D015536:Down-Regulation; D004705:Endocytosis; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000032:analysis / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D015603:Keratinocytes / Q000201:enzymology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009097:Multienzyme Complexes / Q000502:physiology; D010749:Phosphoprotein Phosphatases / Q000502:physiology*; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D017479:Receptors, Platelet-Derived Growth Factor / Q000032:analysis; D026082:Singlet Oxygen / Q000494:pharmacology*
D009097:Multienzyme Complexes; D011518:Proto-Oncogene Proteins; D026082:Singlet Oxygen; D066246:ErbB Receptors; D017479:Receptors, Platelet-Derived Growth Factor; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex