Activation of adenovirus type 2 early region 4 ORF4 cytoplasmic death function by direct binding to Src kinase domain.
Adenovirus type 2 (Ad2) early region 4 ORF4 (E4orf4) triggers a major death pathway that requires its accumulation in cellular membranes and its tyrosine phosphorylation. This program is regulated by Src family kinases and triggers a potent ZVAD (benzyloxycarbonyl-VAD)- and Bcl2-resistant cell death response in human-transformed cells. How E4orf4 deregulates Src-dependent signaling is unknown. Here we provide strong evidence that a physical interaction requiring the kinase domain of Src and the arginine-rich motif of E4orf4 is involved. The Src binding domain of E4orf4 overlaps with, but is distinct from that of the Balpha subunit of protein phosphatase 2A (PP2A-Balpha) and some E4orf4 complexes contain both PP2A and Src. Functional assays using mutant E4orf4 revealed that deregulation of Src signaling, activation of the Jun kinase pathway, and cell blebbing were all critically dependent on Src binding. In contrast, PP2A-Balpha binding per se was not required to engage the Src-dependent death pathway but was more critical for triggering a distinct death activity. Both E4orf4 death activities were manifested within a given cell population, were typified by distinct morphological features, and contributed to overall cell killing, although to different extents in various cell types. We conclude that E4orf4 binding to the Src kinase domain leads to deregulation of Src signaling and plays a crucial role in induction of the cytoplasmic death pathway. Nonetheless, both Src and PP2A enzymes are critical targets of E4orf4 that likely cooperate to trigger E4orf4-induced tumor cell killing and whose relative contributions may vary in function of the cellular background.
Champagne
Claudia
C
Centre de Recherche en Cancérologie de l'Université Laval, L'Hôtel-Dieu de Québec, CHUQ, Québec G1R 2J6, Canada.
Landry
Marie-Claude
MC
Gingras
Marie-Claude
MC
Lavoie
Josée N
JN
D017504:Adenovirus E4 Proteins / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D016923:Cell Death; D002478:Cells, Cultured; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D054648:Protein Phosphatase 2; D015398:Signal Transduction; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*; D018909:src Homology Domains; D019061:src-Family Kinases / Q000378:metabolism*
D017504:Adenovirus E4 Proteins; C121517:E4orf4 protein, adenovirus; D014764:Viral Proteins; D019061:src-Family Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2