Abrogation of interleukin-3 dependence of myeloid cells by the v-src oncogene requires SH2 and SH3 domains which specify activation of STATs

The v-src oncogene encodes a nonreceptor tyrosine kinase, When this gene was expressed in the myeloblastic cell line 32Dc13, 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 The v-src oncogene, 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 32Dc13 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 32Dc13 cell line IL-3 independent, To understand the possible mechanisms underlying the IL-3-independent growth of v-src-transformed 32Dc13 cells, we examined the phosphorylation status of JAK-1, JAK-2, and JAK-3 kinases in the v-src- and v-fgr-transformed 32Dc13 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 32Dc13 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 32Dc13 cells, do not coimmunoprecipitate with v-Src, suggesting that these proteins either interact weakly,vith v-Src or are phosphorylated by a mechanism distinctive from that of STAT3.