Sequences surrounding the Src-homology 3 domain of phospholipase Cgamma-1 increase the domain's association with Cbl.
SH3 domains are protein modules that interact with proline-rich polypeptide fragments. Cbl is an adapter-like protein known to interact with several SH3 domains, including the PLCgamma1 SH3 domain and the Grb2 amino terminal SH3 domain. Here we explore whether sequences surrounding the PLCgamma1 SH3 domain core sequence (aa.796-851) can affect the binding to Cbl, a target used as a prototypical ligand. Consistent with previous reports, our results demonstrated a weak binding of Cbl to GST fusion proteins that strictly encompass the structural core of the PLCgamma1 SH3 domain but a high-avidity binding to the Grb2 amino-terminal SH3 domain. Inclusion of amino acids immediately flanking the PLCgamma1 SH3 core domain, however, substantially increased binding of Cbl to a level comparable to that of the Grb2 amino-terminal SH3 domain. The interaction of this extended PLCgamma1 SH3 domain fusion protein with Cbl was shown to depend entirely upon the interaction of the domain with a proline-rich motif in Cbl, ruling out the possibility that amino acids adjacent to the core SH3 domain of PLCgamma1 provide independent Cbl binding. These data suggest that sequences surrounding the SH3 domain of PLCgamma1 may contribute to or stabilize the association of the domain with the target protein, thus increasing its binding efficiency.
Division of Monoclonal Antibodies, US-Food and Drug Administration, Bethesda, Maryland, 20852, USA.
L J
LJ
Graham
B A
BA
Stoica
M
M
Shapiro
K E
KE
DeBell
B
B
Rellahan
J
J
Laborda
E
E
Bonvini
D001665:Binding Sites; D001667:Binding, Competitive; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D051966:Phospholipase C gamma; D011392:Proline / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D010738:Type C Phospholipases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D018909:src Homology Domains
D007527:Isoenzymes; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D011392:Proline; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; C067419:CBL protein, human