期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2016
卷号：113
期号：48
页码：13732-13737
DOI：10.1073/pnas.1611904113
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceSrc homology 2 (SH2) domains regulate signaling by binding phosphotyrosines. While all known SH2 domains consist of contiguous sequences of [~]100 aa, we describe a highly conserved SH2-like sequence motif located in the second cytoplasmic loop of membrane-spanning domain of erythrocyte membrane band 3. Upon tyrosine phosphorylation of the cytoplasmic domain of band 3, the domain rotates to bind the SH2 signature sequence within the membrane-spanning domain of band 3, promoting significant changes in erythrocyte properties. Because sequence homology searches do not recognize SH2-like sequence motifs, the possibility arises that many other SH2-like structures remain undetected in membrane-spanning proteins where they perform critical steps in kinase-facilitated signaling pathways. Src homology 2 (SH2) domains are composed of weakly conserved sequences of [~]100 aa that bind phosphotyrosines in signaling proteins and thereby mediate intra- and intermolecular protein-protein interactions. In exploring the mechanism whereby tyrosine phosphorylation of the erythrocyte anion transporter, band 3, triggers membrane destabilization, vesiculation, and fragmentation, we discovered a SH2 signature motif positioned between membrane-spanning helices 4 and 5. Evidence that this exposed cytoplasmic sequence contributes to a functional SH2-like domain is provided by observations that: (i) it contains the most conserved sequence of SH2 domains, GSFLVR; (ii) it binds the tyrosine phosphorylated cytoplasmic domain of band 3 (cdb3-PO4) with Kd = 14 nM; (iii) binding of cdb3-PO4 to erythrocyte membranes is inhibited both by antibodies against the SH2 signature sequence and dephosphorylation of cdb3-PO4; (iv) label transfer experiments demonstrate the covalent transfer of photoactivatable biotin from isolated cdb3-PO4 (but not cdb3) to band 3 in erythrocyte membranes; and (v) phosphorylation-induced binding of cdb3-PO4 to the membrane-spanning domain of band 3 in intact cells causes global changes in membrane properties, including (i) displacement of a glycolytic enzyme complex from the membrane, (ii) inhibition of anion transport, and (iii) rupture of the band 3-ankyrin bridge connecting the spectrin-based cytoskeleton to the membrane. Because SH2-like motifs are not retrieved by normal homology searches for SH2 domains, but can be found in many tyrosine kinase-regulated transport proteins using modified search programs, we suggest that related cases of membrane transport proteins containing similar motifs are widespread in nature where they participate in regulation of cell properties.
关键词：SH2 domain motif ; tyrosine phosphorylation ; erythrocyte glycolysis ; anion exchanger 1 ; regulation of transport proteins
