title: Abrogation of interleukin-3 dependence of myeloid cells by the v-src oncogene requires SH2 and SH3 domains which specify activation of STATs.
abstract: The v-src oncogene encodes a nonreceptor tyrosine kinase. When this gene was expressed in the myeloblastic cell line 32Dcl3, it was found to abrogate interleukin-3 (IL-3) dependence of this cell line and to block its ability to terminally differentiate into granulocytes in response to granulocyte colony-stimulating factor (GCSF). In contrast, a highly related tyrosine kinase gene, v-fgr, fails to render this cell line IL-3 independent for growth or to block its ability to undergo terminal differentiation in the presence of GCSF. The active structural domains of v-src that are responsible for the abrogation of IL-3 dependence of myeloid cells and the mechanisms by which v-src transforms these cells are at present unclear. To identify the domains in v-src which are responsible for this activity, we constructed several chimeric recombinants between the v-src and the related Src family member v-fgr by replacing portions of v-src with corresponding domains of v-fgr. These chimeric DNAs were transfected into 32Dcl3 cells and examined for their abilities to render this cell line IL-3 independent. Our results show that only chimeras containing both the SH3 and the SH2 domains of v-src were capable of rendering the 32Dcl3 cell line IL-3 independent. To understand the possible mechanisms underlying the IL-3-independent growth of v-src-transformed 32Dcl3 cells, we examined the phosphorylation status of JAK-1, JAK-2, and JAK-3 kinases in the v-src- and v-fgr-transformed 32Dcl3 cells. Our results show that none of the JAK kinases are constitutively phosphorylated by v-src or v-fgr. We then examined the phosphorylation status of the STAT (signal transducers and activators of transcription) family of transcription factors. Our results show that STAT1, STAT3, and STAT5 exist in a constitutively phosphorylated state in v-src-transformed 32Dcl3 cells, while such constitutive phosphorylation is not seen in v-fgr-transformed cell lines. Our results also show that STAT3 coimmunoprecipitates with v-Src, suggesting that the activation of STAT3 occurs due to direct association with v-Src. However, STAT1 and STAT5, which also exist in a constitutively phosphorylated state in v-src-transformed 32Dcl3 cells, do not coimmunoprecipitate with v-Src, suggesting that these proteins either interact weakly with v-Src or are phosphorylated by a mechanism distinctive from that of STAT3.
authors.affiliation: Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
authors.forename: P
authors.initials: P
authors.lastname: Chaturvedi
authors.forename: S
authors.initials: S
authors.lastname: Sharma
authors.forename: E P
authors.initials: EP
authors.lastname: Reddy
mesh_terms: D000209:Acute-Phase Proteins / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000378:metabolism*; D007377:Interleukin-3 / Q000378:metabolism*; D051379:Mice; D008894:Milk Proteins; D015688:Oncogene Protein pp60(v-src) / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D015682:Retroviridae Proteins, Oncogenic / Q000494:pharmacology; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D050799:STAT5 Transcription Factor; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000378:metabolism*; D018909:src Homology Domains
chemical_list: D000209:Acute-Phase Proteins; D004268:DNA-Binding Proteins; D007377:Interleukin-3; D008894:Milk Proteins; D011993:Recombinant Fusion Proteins; D015682:Retroviridae Proteins, Oncogenic; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D050799:STAT5 Transcription Factor; C494080:Stat1 protein, mouse; C494087:Stat3 protein, mouse; D015534:Trans-Activators; C052601:v-fgr protein, Feline sarcoma virus; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src)