EGF-dependent association of phospholipase C-gamma1 with c-Cbl.
The structure of phospholipase Cgamma1 (PLC-gamma1) contains two SH2 domains and one SH3 domain. While the function of the SH2 domains in PLC-gamma1 are well described, to date no growth factor-dependent function for the SH3 domain has been presented. To assess SH3 domain function in the context of the full-length PLC-gamma1, this domain was deleted and the mutant was stably expressed in Plcg1 null mouse embryonic fibroblasts. Following EGF treatment of cells, the PLC-gamma1DeltaSH3 mutant displayed the same increased level of tyrosine phosphorylation and association with EGF receptor as wild-type PLC-gamma1. Also, the SH3 mutant demonstrated membrane translocation and mediated the mobilization of intracellular Ca(2+) in response to EGF. c-Cbl is shown to associate with tyrosine phosphorylated PLC-gamma1 in an EGF-dependent manner, but no association was detected with the PLC-gamma1DeltaSH3 mutant. Interestingly, PDGF, which also tyrosine phosphorylates PLC-gamma1, failed to induce c-Cbl association with PLC-gamma1 and also provoked no c-Cbl tyrosine phosphorylation. This suggests that c-Cbl tyrosine phosphorylation is necessary for its interaction with PLC-gamma1. Evidence of a direct association of c-Cbl with PLC-gamma1 was provided by pull-down and overlay experiments, using glutathione S-transferase fusion proteins that contain the SH3 domain of PLC-gamma1. The data, therefore, show an EGF-inducible direct association of PLC-gamma1 with c-Cbl in vivo that is mediated by the SH3 domain of PLC-gamma1.
Tvorogov
Denis
D
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Carpenter
Graham
G
D016475:3T3 Cells; D000818:Animals; D000077214:Becaplermin; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000378:metabolism* / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D051966:Phospholipase C gamma; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D020574:Proto-Oncogene Proteins c-sis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D018909:src Homology Domains / Q000502:physiology*
D007527:Isoenzymes; D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D020574:Proto-Oncogene Proteins c-sis; D011993:Recombinant Fusion Proteins; D000077214:Becaplermin; D014443:Tyrosine; D004815:Epidermal Growth Factor; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; C067419:CBL protein, human; C493849:Cbl protein, mouse; D002118:Calcium