A Single Point Mutation Switches the Specificity of Group III Src Homology (SH) 2 Domains to That of Group I SH2 Domains

Abstract

Stimulation of cellular responses by growth factors and cytokines is often accomplished by the activation of protein-tyrosine kinases (1, 6). One of the major consequences of tyrosine phosphorylation is to induce a specific set of protein-protein interactions, and thereby initiate a series of intracellular signaling cascades. The SH2 1 domains of cytosolic signaling proteins mediate the assembly of such complexes by binding to phosphotyrosine moieties within specific sequence contexts (1, 6, 7). Therefore, in order to understand signal transduction by protein-tyrosine kinases, it is important to decode the mechanisms by which SH2 domains achieve specificity in their recognition of phosphotyrosine sites.The specificity of SH2 domains was first systematically studied using a degenerate phosphopeptide library (2, 3). Subsequently, crystal structures of SH2 domains complexed with their high affinity ligands were obtained, providing a structural basis for phosphopeptide recognition by SH2 domains (4,5,8,9). From these studies and related experiments analyzing the in vivo binding sites of various SH2 domains, it has become evident that 3-6 residues C-terminal of phosphotyrosine dictate the specificity of SH2 domain binding. Importantly, SH2 domains can be divided into four subgroups on the basis of their specificity and primary sequences (2). For example, Group I SH2 domains (SH2 domains of Src family, Abl, Crk, GRB2, Nck SH2 domains, etc.) select the general motif Tyr(P)-hydrophilichydrophilic-hydrophobic and have an aromatic amino acid at the ␤D5 position. However, Group III SH2 domains (e.g. SH2 domains of phosphoinositide 3-kinase p85, phospholipase C-␥, and Syp/SHPTP2) select the general motif Tyr(P)-hydrophobic-X-hydrophobic and have Ile or Cys at the ␤D5 position (Fig. 1A). In the three-dimensional structures of Src and Lck SH2 domains, the ␤D5 Tyr is at the surface and makes contacts with the side chains of both the pYϩ1 and pYϩ3 residues of the bound phosphopeptide (4, 5). However, in the three-dimensional structures of Syp and PLC-␥ SH2 domains (Group III), the aliphatic residue at the ␤D5 position is buried deeper in the protein, opening a hydrophobic cavity for the pYϩ1 through pYϩ6 residues (8, 9) (Fig. 1B). To address the importance of the ␤D5 position, we have investigated the effect of substituting this residue on the specificity of two Group III SH2 domains. MATERIALS AND METHODSMutant SH2 Domain Construction-The point mutation (I383Y) on phosphoinositide 3-kinase p85 N-terminal SH2 domain was achieved by PCR mutagenesis using human p85 cDNA as template. The following primers were synthesized: 1, 5Ј-GACGGATCCTTACAAAATGCTGAA-3Ј; 2, 5Ј-GTCGAATTCCATCTTCTTTGACAACTTG-3Ј; 3, 5Ј-GGAAAT-AACAAATTATACAAAATATTTCATCGA-3Ј; 4, 5Ј-TCGATGAAATAT-TTTGTATAATTTGTTATTTCC-3Ј. Two PCR fragments were first produced using primer pairs 1 and 4, and 2 and 3. The mutant cDNA was obtained by ligating these two fragments using PCR with primers 1 and 2. The final PCR products were cut with BamHI and EcoRI, and cloned into pGEX2T v...

Functional Diversity of Csk, Chk, and Src SH2 Domains due to a SingleResidueVariation

“…This result also demonstrates how easily SH2 domains with new properties could be evolved. Two cases in which a single point mutation could switch the binding specificity have been reported (46,47), but two homologous SH2 domains possessing dramatically different binding preferences due to the difference in one residue has not been previously reported to our knowledge.…”

Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site

“…In some cases, peptide studies may not provide accurate estimates of SH2 binding speci®city since synthetic peptides may not mimic all the structural features of SH2 binding sites on autophosphorylated receptors. In fact, this may be particularly true for PLCg and src family members where it has been shown that binding is not determined solely by the three amino acids carboxy-terminal to the phosphorylated tyrosine (Alonso et al, 1995;Songyang et al, 1995;Pascal et al, 1994;Larose et al, 1995). The optimal sequence carboxy-terminal to phosphotyrosine that binds to the src SH2 domain has been determined from a library of phosphopeptides and was found to be pYEEI.…”

“…The optimal sequence carboxy-terminal to phosphotyrosine that binds to the src SH2 domain has been determined from a library of phosphopeptides and was found to be pYEEI. The selectivity of the src SH2 domain was highest for a large aliphatic amino acid at position +3, with slightly lower selections at positions +1 and +2 (Songyang et al, 1995). The residues carboxy-terminal to tyrosine 821 (EiLpYVNMD) of the Ufo receptor, match this sequence at the +2 and +3 positions.…”

Specificity and affinity motifs for Grb2 SH2-ligand interactions

“…All known SH2 domains can be placed into four different groups on the basis of the amino acid at position ␤D5. The side chain at the ␤D5 position contacts pϩ1 and pϩ3 of the phospho-tyrosine ligand and has been shown to have a major effect on SH2 selectivity (3,24,25). In our set of control SH2 domains we included three closely related SH2 domains from signaling molecules Abl, Src, and Nck that are derived from the same group as Grb2 (group 1).…”