The receptors for human interleukin-3(IL-3) and human granulocyte-macrophage colony-stimulating factor (GM-CSF), hIL-3R, hGM-CSFR, respectively, consists of two subunits, ⍺ and β, both of which are members of the cytokine receptor superfamily. Phosphorylation of tyrosine residues in the hGMR β subunit and several cellular proteins is observed after hGM-CSF stimulation. We analyzed the role of tyrosine residues in the hGMR β subunit and the nature of tyrosine kinase, JAK2, in hGMR signal transduction using several hGMR β subunit mutants. In addition to the box1 region, a membrane distal region (a.a. 544-589) of the hGMRβ was required for c-fos activation. Only one tyrosine residue (Tyr577) existed within the region 544 to 589, and substitution of Tyr577 to phenylalanine in GMR β 589 resulted in loss of c-fos activation. In contrast, the same substitution in a wild type receptor did not affect GM-CSF induced activities such as c-fos messenger RNA (mRNA) induction and proliferation, but the substitution abolished Shc phosphorylation. These results suggest that the activation of Shc is not essential for c-fos activation and several tyrosine residues cooperate for c-fos activation. It is well documented that IL-3 or GM-CSF activate JAK2 in BA/F3 cells. The role of JAK2 in IL-3/GM-CSF functions, however, is largely unknown. We examined the role of JAK2 in GM-CSF induced signaling pathways. Dominant negative JAK2 (ΔJAK2) lacking the C-terminus kinase domain suppressed IL-3/GM-CSF induced c-fos activation and c-myc activation and proliferation, suggesting that JAK2 was involved in both signaling pathways. Protein tyrosine phosphatase SHP-2 (also called PTP 1D) and Shc were phosphorylated by IL-3/GM-CSF in BA/F3 cells; however, these phosphorylation events were inhibited by the expression of ΔJAK2. Taken together, these results indicate that JAK2 is a primary kinase regulating all the known activities of GM-CSF. JAK2 mediates GM-CSF induced c-fos activation through receptor phosphorylation and Shc/PTP 1D activation. (J Allergy Clin Immunol 1996;98:S183-91.)