TAILIEUCHUNG - Báo cáo khoa học: Single phosphorylation of Tyr304 in the cytoplasmic tail of ephrin B2 confers high-affinity and bifunctional binding to both the SH2 domain of Grb4 and the PDZ domain of the PDZ-RGS3 protein

TheB class cell-attachedephrinsmediate contact-dependent cell–cell communications and transduce the contact signals to the host cells through the binding interactions of their cytoplasmic domains. Two classes of intracellular effectors of B ephrins have been identified: one contains the PSD-95/ Dlg/ZO-1 (PDZ)domain(for examplePDZ-RGS3), and the second the Src homology 2 (SH2) domain (. the Grb4 adaptor protein). The interaction with Grb4 requires phos-phorylation of tyrosine residues on the conserved cytoplas-mic C-terminal region of B ephrins, while binding to the PDZ domain is independent of tyrosine phosphorylation | Eur. J. Biochem. 271 1725-1736 2004 FEBS 2004 doi Single phosphorylation of Tyr304 in the cytoplasmic tail of ephrin B2 confers high-affinity and bifunctional binding to both the SH2 domain of Grb4 and the PDZ domain of the PDZ-RGS3 protein Zhengding Su Ping Xu and Feng Ni Biomolecular NMR and Protein Research Group Biotechnology Research Institute National Research Council of Canada Montreal Quebec Canada The B class cell-attached ephrins mediate contact-dependent cell-cell communications and transduce the contact signals to the host cells through the binding interactions of their cytoplasmic domains. Two classes of intracellular effectors of B ephrins have been identified one contains the PSD-95 Dlg ZO-1 PDZ domain for example PDZ-RGS3 and the second the Src homology 2 SH2 domain . the Grb4 adaptor protein . The interaction with Grb4 requires phosphorylation of tyrosine residues on the conserved cytoplasmic C-terminal region of B ephrins while binding to the PDZ domain is independent of tyrosine phosphorylation. However the exact phosphorylation site s required for signaling remained obscure and it is also unknown whether the two classes of effectors can bind to B ephrins simultaneously or if the binding of one affects the binding of the other. We report here that phosphorylation of Tyr304 in the functional C-terminal region residues 301-333 of ephrin B2 confers high-affinity binding to the SH2 domain of the Grb4 protein. Tyrosine phosphorylation at other candidate sites resulted in only minor change of the binding of Tyr304-phosphorylated ephrin B peptide . eph-rinB2 301-333 -pY304 with the SH2 domain. 1H-15N NMR HSQC experiments show that only the eph-rinB2 301-333 -pY304 peptide forms a stable and specific binding complex with the SH2 domain of Grb4. The SH2 and PDZ domains were found to bind to the Tyr304 phosphopeptide both independently and at the same time forming a three-component molecular complex. Taken together .

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