Band 3 is an anchor protein and a target for SHP-2 tyrosine phosphatase in human erythrocytes.
Tyr phosphorylation of the multifunctional transmembrane protein band 3 has been implicated in several erythrocyte functions and disorders. We previously demonstrated that pervanadate treatment of human erythrocytes induces band-3 Tyr phosphorylation, which is catalyzed by the sequential action of tyrosine kinase Syk and tyrosine kinase(s) belonging to the Src family. In this study, we show that Tyr phosphorylation of band 3, elicited by pervanadate, N-ethylmaleimide, or diamide, greatly increases band-3 interaction with the tyrosine phosphatase SHP-2 in parallel with the translocation of SHP-2 to erythrocyte membranes. These events seem to be mediated by Src-like catalyzed phosphorylation of band 3 because both SHP-2 translocation to cellular membranes and its interaction with Tyr-phosphorylated protein are greatly counteracted by PP2, a specific inhibitor of Src kinases. Binding-competition experiments demonstrate that SHP-2 recruitment to band 3 occurs via its SH2 domain(s). In particular, our data support the view that SHP-2 docks specifically with P-Y359 of band 3. Experiments performed with intact erythrocytes in the presence of the SHP-2 inhibitor calpeptin suggest that, once recruited to Tyr-phosphorylated band 3, the tyrosine phosphatase dephosphorylates the protein. P-Y8, 21, and 904 are the residues affected by SHP-2, as judged by (32)P-peptide mapping of band 3 digested with trypsin. These results indicate that in treated erythrocytes, recruitment of cytosolic SHP-2 to band 3 is a prerequisite for the subsequent dephosphorylation of the transmembrane protein.
Bordin
Luciana
L
Dipartimento di Chimica Biologica, and Centro di Studio delle Biomembrane del Consiglio Nazionale delle Ricerche, University of Padova, Italy.
Brunati
Anna Maria
AM
Donella-Deana
Arianna
A
Baggio
Bruno
B
Toninello
Antonio
A
Clari
Giulio
G
D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D021381:Protein Transport; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases
D001457:Anion Exchange Protein 1, Erythrocyte; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; C516724:PTPN11 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases