Tyrosines 303/343/353 within the Sprouty-related domain of Spred2 are essential for its interaction with p85 and inhibitory effect on Ras/ERK activation.
Sprouty-related EVH1 domain (Spred) proteins modulate growth factor receptor signaling by inhibiting the Ras/ERK pathway. In particular, the Sprouty-related domain (SPR) of Spred2 is essential for the Spred2-mediated inhibitory effect, but the molecular mechanism is largely unknown. We show here that the p85 subunit of phosphatidylinositol 3-kinase (PI3K) is a new binding partner of Spred2 via interaction with the SPR domain. Mutation of three tyrosines 303/343/353 within the SPR domain not only abolish EGF-induced p85 binding to Spred2 but also attenuate the inhibitory effect on Ras/ERK activation by Spred2. This results in increased Hela cell proliferation and neurite outgrowth in PC12 cells. We further demonstrate that p85 binding to Spred2 enhances the Spred2-mediated inhibitory effect via increased Ras binding to Spred2 and decreased Spred2 ubiquitination. We also show that Spred2 constitutively associates with epidermal growth factor receptor (EGFR) via its SPR domain and dissociates from EGFR upon EGF stimulation. Moreover, mutation of tyrosines 303/343/353 together enhances Spred2 binding to EGFR. Taken together, these results suggest critical roles of the three tyrosines 303/343/353 within the SPR domain in regulating Spred2 signaling and provide a mechanism for the SPR domain of Spred2 to mediate the inhibitory effect on the Ras/ERK pathway.
College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, China. ssmeng@yzu.edu.cn
Songshu
S
Meng
Miao
M
Zhang
Wenyan
W
Pan
Zhongliang
Z
Li
Deborah H
DH
Anderson
Shengping
S
Zhang
Baoxue
B
Ge
Chuangui
C
Wang
D000818:Animals; D001665:Binding Sites; D049109:Cell Proliferation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects* / Q000235:genetics; D009154:Mutation; D016501:Neurites / Q000187:drug effects; D016716:PC12 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination / Q000187:drug effects; D018631:ras Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics
D021122:Protein Subunits; D012097:Repressor Proteins; C500760:SPRED2 protein, human; D014443:Tyrosine; D004815:Epidermal Growth Factor; D019869:Phosphatidylinositol 3-Kinases; C512478:EGFR protein, human; D066246:ErbB Receptors; D018631:ras Proteins