Binding and function of phosphotyrosines of the Ephrin A2 (EphA2) receptor using synthetic sterile α motif (SAM) domains.
The sterile α motif (SAM) domain of the ephrin receptor tyrosine kinase, EphA2, undergoes tyrosine phosphorylation, but the effect of phosphorylation on the structure and interactions of the receptor is unknown. Studies to address these questions have been hindered by the difficulty of obtaining site-specifically phosphorylated proteins in adequate amounts. Here, we describe the use of chemically synthesized and specifically modified domain-length peptides to study the behavior of phosphorylated EphA2 SAM domains. We show that tyrosine phosphorylation of any of the three tyrosines, Tyr(921), Tyr(930), and Tyr(960), has a surprisingly small effect on the EphA2 SAM structure and stability. However, phosphorylation at Tyr(921) and Tyr(930) enables differential binding to the Src homology 2 domain of the adaptor protein Grb7, which we propose will lead to distinct functional outcomes. Setting up different signaling platforms defined by selective interactions with adaptor proteins thus adds another level of regulation to EphA2 signaling.
From the Departments of Physiology and Biophysics.
Susmita
S
Borthakur
From the Departments of Physiology and Biophysics.
HyeongJu
H
Lee
From the Departments of Physiology and Biophysics.
SoonJeung
S
Kim
From the Departments of Physiology and Biophysics, Pharmacology, and the Rammelkamp Center for Research, MetroHealth Medical Center, Cleveland, Ohio 44109the Case Comprehensive Cancer Center, and.
Bing-Cheng
BC
Wang
From the Departments of Physiology and Biophysics, the Case Comprehensive Cancer Center, and Neurosciences, the Case Center for Proteomics and Bioinformatics, Case Western Reserve University, Cleveland, Ohio 44106 and matthias.buck@case.edu.
Matthias
M
Buck
D020816:Amino Acid Motifs; D051382:GRB7 Adaptor Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019000:Phosphotyrosine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D036104:Receptor, EphA2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
C495955:GRB7 protein, human; D051382:GRB7 Adaptor Protein; D019000:Phosphotyrosine; D036104:Receptor, EphA2
Adaptor Protein; Biophysics; Endocytosis; Ephrin Receptors; Phosphorylation; Protein-tyrosine Kinase (Tyrosine Kinase); Receptor Clustering and Signaling; SAM Domains; SHIP2