A role of the SAM domain in EphA2 receptor activation.
Among the 20 subfamilies of protein receptor tyrosine kinases (RTKs), Eph receptors are unique in possessing a sterile alpha motif (SAM domain) at their C-terminal ends. However, the functions of SAM domains in Eph receptors remain elusive. Here we report on a combined cell biology and quantitative fluorescence study to investigate the role of the SAM domain in EphA2 function. We observed elevated tyrosine autophosphorylation levels upon deletion of the EphA2 SAM domain (EphA2ΔS) in DU145 and PC3 prostate cancer cells and a skin tumor cell line derived from EphA1/A2 knockout mice. These results suggest that SAM domain deletion induced constitutive activation of EphA2 kinase activity. In order to explain these effects, we applied fluorescence correlation spectroscopy to investigate the lateral molecular organization of EphA2. Our results indicate that SAM domain deletion (EphA2ΔS-GFP) increases oligomerization compared to the full length receptor (EphA2FL-GFP). Stimulation with ephrinA1, a ligand for EphA2, induced further oligomerization and activation of EphA2FL-GFP. The SAM domain deletion mutant, EphA2ΔS-GFP, also underwent further oligomerization upon ephrinA1 stimulation, but the oligomers were larger than those observed for EphA2FL-GFP. Based on these results, we conclude that the EphA2 SAM domain inhibits kinase activity by reducing receptor oligomerization.
Department of Chemistry, University of Akron, Akron, OH, 44325, USA.
Xiaojun
X
Shi
Departments of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, 44106, USA.|Pharmacology, Case Western Reserve University, Cleveland, OH, 44106, USA.|Rammelkamp Center for Research, MetroHealth Medical Center, Cleveland, OH, 44109, USA.
Vera
V
Hapiak
Departments of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, 44106, USA.|Pharmacology, Case Western Reserve University, Cleveland, OH, 44106, USA.|Rammelkamp Center for Research, MetroHealth Medical Center, Cleveland, OH, 44109, USA.
Ji
J
Zheng
Departments of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, 44106, USA.
Jeannine
J
Muller-Greven
Department of Chemistry, University of Akron, Akron, OH, 44325, USA.
Deanna
D
Bowman
Department of Biology, University of Akron, Akron, OH, 44325, USA.
Ryan
R
Lingerak
Departments of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, 44106, USA.|Pharmacology, Case Western Reserve University, Cleveland, OH, 44106, USA.|Department of Neurosciences, Case Western Reserve University, Cleveland, Ohio, 44106, USA.
Matthias
M
Buck
Departments of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, 44106, USA.|Pharmacology, Case Western Reserve University, Cleveland, OH, 44106, USA.|Rammelkamp Center for Research, MetroHealth Medical Center, Cleveland, OH, 44109, USA.
Bing-Cheng
BC
Wang
Department of Chemistry, University of Akron, Akron, OH, 44325, USA.
Adam W
AW
Smith
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D045744:Cell Line, Tumor; D036382:Ephrin-A1 / Q000737:chemistry / Q000378:metabolism; D036383:Ephrin-A2 / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D051379:Mice; D010766:Phosphorylation; D011485:Protein Binding; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D036104:Receptor, EphA2; D000071682:Sterile Alpha Motif
C000623258:EPHA2 protein, human; D036382:Ephrin-A1; D036383:Ephrin-A2; D036104:Receptor, EphA2