NMR structure of phospho-tyrosine signaling complexes

Abstract

A structural basis for activation and substrate specificity of src tyrosine kinases, and regulation of protein–protein association by tyrosine phosphorylation is described. Lyn, a src‐family tyrosine kinase, recognizes and phosphorylates the immunoreceptor tyrosine‐based activation motif, ITAM, a critical component in transmembrane signal transduction in hemopoietic cells. The structure of an ITAM peptide substrate bound to an active form of Lyn tyrosine kinase was determined by high‐resolution NMR, and a model of the complex was generated using the crystallographic structure of Lck, a closely related Src‐family kinase. The results provide a rationale for the conserved ITAM residues and specificity of Lyn, and suggest that substrate plays a role in stabilizing the kinase conformation optimal for catalysis. It is our hope that the Lck‐ITAM peptide model complex will be useful in aiding structure‐based drug design efforts that target substrate binding determinants in the design. Concerning the regulation of protein–protein association, we report on a complex between erythrocyte band 3 and two glycolytic enzymes, aldolase and glyceraldehyde‐3‐phosphate dehydrogenase. The formation of this complex is negatively regulated by tyrosine phosphorylation of band 3 by p72syk tyrosine kinase. In red blood cells, this association results in a decrease in glycolysis due to competitive inhibition of the glycolytic enzymes. The structure of band 3 recognized by the glycolytic enzymes was determined by solution NMR, and found to be a loop structure with tyrosine centrally positioned and excluded from intermolecular contact. This phosphorylation sensitive interaction, or PSI, loop may be the basis of a general mechanism for negative regulation through tyrosine phosphorylation. © 1999 John Wiley & Sons, Inc. Med Res Rev, 19, No. 4, 295–305, 1999.

Tonic B‐cell and viral ITAM signaling: context is everything

“…This low‐affinity interaction occurs through the amino‐terminal region of Src kinases independently of ITAM phosphorylation and is distinct from SH2‐mediated binding of Src kinases to phosphorylated ITAMs (6–8). As initial ITAM phosphorylation is thought to occur at the amino‐terminal tyrosine (9), support also comes from structural studies that indicate that side chains of the hydrophilic residue upstream of the amino‐terminal tyrosine of the immunoglobulin α (Igα) ITAM make several hydrogen bonds with the Src family kinase member Lyn (10). The importance of this interaction was demonstrated upon mutation of this hydrophilic residue, which abolished Lyn binding in an enzyme assay (11).…”

“…Additionally, phage display libraries have demonstrated that Src family kinase members have different preferred substrates (26). While Lyn was shown to require the upstream D/E of Igα(10), other members of the Src kinase family may have different or less stringent requirements for this upstream residue, a possibility that remains to be formally tested.…”

Structural basis for the activity of pp60c-srcprotein tyrosine kinase inhibitors

“…As mentioned above, there are no crystal structures of Src or Src family kinases with peptide substrate bound to the active site. However, Post et al52 have determined the substrate conformation of the ITAM peptide (DENLYEGLNLDD) bound to the Src family PTK, Lyn, using transferred nuclear Overhauser effect (NOE). In this structure, tyrosine is positioned next to Asp 181 (c‐src numbering).…”