Raf activation is regulated by tyrosine 510 phosphorylation in Drosophila.
The proto-oncoprotein Raf is pivotal for mitogen-activated protein kinase (MAPK) signaling, and its aberrant activation has been implicated in multiple human cancers. However, the precise molecular mechanism of Raf activation, especially for B-Raf, remains unresolved. By genetic and biochemical studies, we demonstrate that phosphorylation of tyrosine 510 is essential for activation of Drosophila Raf (Draf), which is an ortholog of mammalian B-Raf. Y510 of Draf is phosphorylated by the c-src homolog Src64B. Acidic substitution of Y510 promotes and phenylalanine substitution impairs Draf activation without affecting its enzymatic activity, suggesting that Y510 plays a purely regulatory role. We further show that Y510 regulates Draf activation by affecting the autoinhibitory interaction between the N- and C-terminal fragments of the protein. Finally, we show that Src64B is required for Draf activation in several developmental processes. Together, these results suggest a novel mechanism of Raf activation via Src-mediated tyrosine phosphorylation. Since Y510 is a conserved residue in the kinase domain of all Raf proteins, this mechanism is likely evolutionarily conserved.
Xia
Fan
F
Department of Biomedical Genetics, University of Rochester Medical Center, Rochester, New York, United States of America.
Li
Jinghong
J
Hickey
Gavin W
GW
Tsurumi
Amy
A
Larson
Kimberly
K
Guo
Dongdong
D
Yan
Shian-Jang
SJ
Silver-Morse
Louis
L
Li
Willis X
WX
D000818:Animals; D002460:Cell Line; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000201:enzymology / Q000378:metabolism*; D004789:Enzyme Activation; D018507:Gene Expression Regulation, Developmental; D018698:Glutamic Acid / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D048490:raf Kinases / Q000235:genetics / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism
D029721:Drosophila Proteins; D011518:Proto-Oncogene Proteins; D019000:Phosphotyrosine; D018698:Glutamic Acid; D011505:Protein-Tyrosine Kinases; C075699:Src64B protein, Drosophila; D048490:raf Kinases; C417283:Ras85D protein, Drosophila; D018631:ras Proteins