Constitutive association of EGF receptor with the CrkII-23 mutant that inhibits transformation of NRK cells by EGF and TGF-beta.
Crk belongs to the adapter proteins that participate in many signalling pathways from cell surface receptors. We have characterised the CrkII-23 mutant that inhibits the transformation of NRK cells induced by epidermal growth factor (EGF) and transforming growth factor (TGF)-beta. To study the biochemical difference, cDNAs of the wild-type CrkII and the CrkII-23 mutant were introduced stably into NIH 3T3 cells expressing EGF receptor (EGFR). Both CrkII and CrkII-23 were phosphorylated on tyrosine upon EGF simulation with similar time course and dose dependency. Whereas the wild-type CrkII bound to EGFR only after EGF stimulation, CrkII-23 bound to EGFR from before stimulation. Mutation in the Src homology (SH) 2 or amino-terminal SH3 domain did not abolish the binding of CrkII-23 to EGFR in the quiescent cells, suggesting that the binding is mediated by a novel mechanism. These CrkII-23-derived mutants, however, did not suppress transformation of NRK cells by EGF and TGF-beta. Hence, both the SH2 and amino-terminal SH3 domains are required to inhibit transformation of NRK cells. These results suggest that persistent signalling from CrkII-23 bound to EGFR suppresses transformation by EGF and TGF-beta in NRK23 cells.
Ota
S
S
Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan.
Kizaka-Kondoh
S
S
Hashimoto
Y
Y
Nishihara
H
H
Nagashima
K
K
Kurata
T
T
Okayama
H
H
Matsuda
M
M
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D019556:COS Cells; D002454:Cell Differentiation / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000378:metabolism*; D015852:Eukaryotic Initiation Factor-2; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D051379:Mice; D009154:Mutation; D009474:Neurons / Q000166:cytology / Q000187:drug effects; D016716:PC12 Cells; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D016315:Proto-Oncogene Proteins c-abl / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl; D050737:Proto-Oncogene Proteins c-crk; D051381:Rats; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D018909:src Homology Domains / Q000235:genetics*
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; C493872:Crk protein, rat; D015852:Eukaryotic Initiation Factor-2; D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; C528035:Shc1 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D016212:Transforming Growth Factor beta; D014443:Tyrosine; D004815:Epidermal Growth Factor; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D011494:Protein Kinases; D066246:ErbB Receptors; D016315:Proto-Oncogene Proteins c-abl; C067419:CBL protein, human; C493849:Cbl protein, mouse