CrkL mediates Ras-dependent activation of the Raf/ERK pathway through the guanine nucleotide exchange factor C3G in hematopoietic cells stimulated with erythropoietin or interleukin-3.
CrkL is an SH2 and SH3 domain-containing adaptor protein implicated in pathogenesis of chronic myelogenous leukemia. Here, we demonstrate that overexpression of CrkL enhances the erythropoietin (Epo)- or interleukin (IL)-3-induced activation of Elk-1 and the c-fos gene promoter activity in 32D/EpoR-Wt cells. Moreover, the Epo-induced activation of ERK1 and ERK2 was augmented and prolonged in cells inducibly overexpressing CrkL. A moderate increase in Epo-induced activation of JNK was also observed in cells overexpressing CrkL. Overexpression of C3G enhanced the Elk-1 activation synergistically with CrkL, while a C3G mutant lacking the guanine nucleotide exchange domain showed an inhibitory effect. Studies using a dominant negative Ha-Ras mutant demonstrated that the Elk-1 and ERK2 activation enhanced by CrkL and C3G was dependent on Ras. Consistent with this, the Epo-induced activation of Ras was augmented in cells inducibly overexpressing CrkL. Most importantly, a CrkL mutant defective in the SH2 or N-terminal SH3 domain showed an inhibitory effect on the Epo-induced activation of ERK2. These data indicate that the CrkL-C3G complex plays a role in Epo- or IL-3-induced, Ras-dependent activation of the Raf/ERK pathway leading to the activation of Elk-1 and the c-fos gene transcription.
Nosaka
Y
Y
First Department of Internal Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan.
Arai
A
A
Miyasaka
N
N
Miura
O
O
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001854:Bone Marrow Cells / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D004921:Erythropoietin / Q000494:pharmacology*; D016762:Genes, fos; D020735:Guanine Nucleotide-Releasing Factor 2 / Q000378:metabolism*; D007377:Interleukin-3 / Q000494:pharmacology*; D020935:MAP Kinase Signaling System; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; D018631:ras Proteins / Q000378:metabolism*; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; C082869:CRKL protein; D004268:DNA-Binding Proteins; C494113:Elk1 protein, mouse; D020735:Guanine Nucleotide-Releasing Factor 2; D007377:Interleukin-3; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; D004921:Erythropoietin; D018631:ras Proteins