Somatic JAK1 mutations facilitate the activation of downstream signaling pathways. (A) Autophosphorylation of mutant JAK1 proteins. Mutant, but not wild-type, JAK1 proteins are activated. (B) Growth of Ba/F3 cells expressing JAK1 and mutants in the absence of IL-3. Both mutant and wild-type JAK1-expressing cells can grow in the absence of IL-3. (C) Mutant JAK1 proteins do not affect sensitivity of Ba/F3 cells to IL-3. Cells expressing wild-type or JAK1 mutants were plated in different concentrations of IL-3, and cell growth was measured by MTT assay. The V623A mutant was mildly resistant to high doses of IL-3. Error bars represent SD. (D) Activation of downstream signaling pathways by JAK1 mutants. Protein lysates of Ba/F3 cells expressing wild type and JAK1 mutants were analyzed by Western blot using phospho-specific antibodies shown. Stat1, Stat3, Akt, and Erk signaling was activated in cells expressing each JAK1 mutation. (E) Activation of STAT1 by interferon α in cells expressing JAK1^WT, JAK1^T478S, and JAK1^V623A. STAT1 phosphorylation 15 minutes after stimulation was consistently increased in cells expressing JAK1^T478S and JAK1^V623A compared with JAK1^WT. (F) Densitometry of results in panel E showing increased STAT1 phosphorylation in JAK1 mutant-expressing cells. U4A parental cells that do not express JAK1 are shown as controls. Similar results were obtained in three independent experiments, and a representative example is shown.