Regulation of JAK3 expression and activation in human B cells and B cell malignancies.

Members of the Janus family (JAK) of protein tyrosine kinases are critical enzymes in signaling pathways via hematopoietin receptors. We have cloned JAK3, which unlike other known family members (JAK1, JAK2, and TYK2) is preferentially expressed in hematopoietic cells but not in a variety of other cells. Functionally, JAK3 and JAK1 are coupled to the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 in T cells and NK cells. Because of the importance of IL-2, IL-4, and IL-7 in B cell physiology, we sought to determine whether JAK3 was also present in B lymphocytes and whether it was involved in signaling via cytokines that are important for B cell development and function. In this report, we demonstrate that JAK3 is expressed in normal human peripheral blood B cells at levels that are comparable to those in T cells. In addition, the levels were found to be markedly up-regulated following stimulation with staphylococcal protein A Cowan and anti-CD40 Abs. In addition, IL-4 and IL-7 induced the rapid tyrosine phosphorylation of JAK3 and JAK1, and IL-4 activated both JAK3 and JAK1 phosphotransferase activity. JAK3 protein was also detected in immature B cell lines, but not in more well differentiated cell lines. Additionally, JAK3 was detected in lysates from bone marrow lymphoblasts of patients with B cell precursor acute lymphocytic leukemia and cell lines derived from human B cell lymphomas. Together, these data suggest that the regulation of JAK3 expression and activity is likely to be important in B cell development and function.

Thrombopoietin (TPO) induces tyrosine phosphorylation and activation of STAT5 and STAT3.

The growth and differentiation of megakaryocytes are regulated by thrombopoietin (TPO), a recently characterized cytokine which exerts its effects via a member of the hematopoietin receptor superfamily, c-Mpl. Since many cytokines which bind hematopoietin receptors activate the STAT family of transcription factors, we investigated whether STAT proteins were activated by TPO. TPO induced the formation of a DNA-binding complex recognizing a known STAT-binding sequence. STAT5 was a major component of this DNA-binding complex, and STAT5 was tyrosine phosphorylated in response to TPO. Additionally, TPO-induced the tyrosine phosphorylation and DNA-binding activity of STAT3. Together with the recent demonstration of JAK2 activation in response to TPO, the data presented here define a rapid signaling pathway likely to be important in TPO-induced gene regulation.

Similarities and differences in signal transduction by interleukin 4 and interleukin 13: analysis of Janus kinase activation.

The cytokines interleukin (IL) 4 and IL-13 induce many of the same biological responses, including class switching to IgE and induction of major histocompatibility complex class II antigens and CD23 on human B cells. It has recently been shown that IL-4 induces the tyrosine phosphorylation of a 170-kDa protein, a substrate called 4PS, and of the Janus kinase (JAK) family members JAK1 and JAK3. Because IL-13 has many functional effects similar to those of IL-4, we compared the ability of IL-4 and IL-13 to activate these signaling molecules in the human multifactor-dependent cell line TF-1. In this report we demonstrate that both IL-4 and IL-13 induced the tyrosine phosphorylation of 4PS and JAK1. Interestingly, although IL-4 induced the tyrosine phosphorylation of JAK3, we did not detect JAK3 phosphorylation in response to IL-13. These data suggest that IL-4 and IL-13 signal in similar ways via the activation of JAK1 and 4PS. However, our data further indicate that there are significant differences because IL-13 does not activate JAK3.

Thrombopoietin induces tyrosine phosphorylation and activation of the Janus kinase, JAK2.

Thrombopoietin (TPO) is a recently characterized growth and differentiation factor for megakaryocytes and platelets that exerts its effects via the receptor, c-MpI. This receptor is a member of the hematopoietin receptor superfamily and is essential for megakaryocyte maturation; however, the molecular mechanisms of TPO and c-MpI action have not been elucidated. Recently, the Janus kinases have emerged as important elements in signaling via this family of receptors. In this report, we show that, in the M07e megakaryocytic cell line, which expresses c-MpI and proliferates in response to TPO, TPO induces phosphorylation of a number of substrates between 80 and 140 kD. Specifically, we show that stimulation with TPO induces the rapid tyrosine phosphorylation of a 130-kD protein that we identify as the Janus kinase, JAK2. However, no detectable tyrosine phosphorylation of JAK1, JAK3, or TYK2 was observed. TPO also induced activation of JAK2 phosphotransferase activity in vitro. Taken together, these data indicate that JAK2 likely plays a key role in TPO-mediated signal transduction.