Requirement of multiple SH3 domains of Nck for ligand binding.
The Nck adaptor protein comprises a single C-terminal SH2 domain and three SH3 domains. The domain structure of Nck suggests that Nck links tyrosine kinase substrates to proteins containing proline-rich motifs. Here we show that Bcr/Abl tyrosine kinase, and three tyrosine phosphorylated proteins (115, 120 and 155 kDa) are co-immunoprecipitated with antibody against Nck from lysates of the human leukaemia cell line K562. By means of affinity purification with the Nck-binding phosphopeptide EPGPY(P)AQPSV, we could also detect the association of endogenous Nck with the proto-oncogene product Cbl. An investigation of the nature of interactions revealed that Bcr/Abl, Cbl, and the 155-kDa tyrosine phosphotyrosine bind exclusively to the SH3 domains of Nck. In addition, none of the single SH3 domains of Nck expressed as glutathione-S-transferase (GST) fusion proteins is able to interact with the proline-rich ligands. However, combined first and second SH3 domains have the capacity to bind Bcr/Abl, Chl and p155. Mutations of conserved tryptophan to Lysine in either of the combined first and second SH3 domains completely abolish ligand binding. These data suggest that cooperation exists among the SH3 domains of Nck for a high-affinity binding of proteins containing proline-rich motifs.
Wunderlich
L
L
Department of Medical Chemistry, Semmelweis University Medical School, Budapest, Hungary.
Gohér
A
A
Faragó
A
A
Downward
J
J
Buday
L
L
D048868:Adaptor Proteins, Signal Transducing; D001665:Binding Sites; D016044:Fusion Proteins, bcr-abl / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D008024:Ligands; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D015513:Oncogene Proteins / Q000378:metabolism*; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D008024:Ligands; C000715470:MAS1 protein, human; C078332:Nck protein; D015513:Oncogene Proteins; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D014364:Tryptophan; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D016044:Fusion Proteins, bcr-abl; C067419:CBL protein, human; D008239:Lysine