A Component of a Heterodimeric Receptor Can Functionally Replace a Homodimeric Receptor in Supporting the Activation and Transforming Activity of JAK2-V617F

Abstract

We have recently identified IL27Ra as a gene from an AML patient that transforms hematopoietic cells to a state of cytokine-independent growth. Importantly, we have found that the gene product of IL27Ra, intereukin-27 receptor (IL27R), is expressed on the cell surface of the leukemic cells of AML patients. IL27R, also know as TCCR and WSX-1, is a component if a heterodimeric type I cytokine receptor that functions as the ligand-binding subunit of the receptor for IL-27. Normally, IL27R functions with the gp130 co-receptor to induce signal transduction in response to IL-27. However, we have demonstrated that IL27R can induce the transformation of hematopoietic cells in a ligand- and gp130-independent manner. IL27R, which contains a JAK-binding Box 1 motif, requires JAK family kinase activity to transform cells. In a potential mechanism of IL27R-mediated transformation, IL27R may function as a homodimer to activate JAK family members. In an effort to determine if IL27R can function as a homodimer, we tested to see if IL27R could activate the V617F mutation of JAK2 commonly found in various myeloproliferative neoplasms (MPNs). It is believed that JAK2-V617F utilizes a homodimeric cytokine receptor complex as a scaffold in order to multimerize and become fully activated via transphosphorylation. Expression of IL27R activated the kinase activity of JAK2-V617F, suggesting that IL27R may form homodimers in cells. In addition, co-expression of IL27R and JAK2-V617F led to transformation of BaF3 cells to cytokine-independence, whereas IL27R expressed with wildtype JAK2 or JAK2-V617F expressed alone did not induce rapid cytokine-independent growth of these cells. This is similar data to that obtained with co-expression of JAK2-V617F with homodimeric receptors (e.g. EpoR, GCSFR), demonstrating that a component of a heterodimeric cytokine receptor can functionally replace a homodimeric receptor in mediating JAK2-V617F activation. Chemical crosslinking showed that IL27R could dimerize suggesting that IL27R may form a scaffold for JAK2-V617F-mediated transformation. In addition to providing a scaffold for binding and activation of JAK2-V617F, it has been proposed that homodimeric receptors also provide an important substrate of the JAK2 tyrosine kinase activity to induce cell signaling and transformation. Tyrosine phosphorylation of the scaffolding receptor presumably recruits signaling molecules (e.g. STATs) to the receptor where they can be activated by JAK2 phosphorylation. We have mutated the two tyrosine residues in the cytoplasmic domain of IL27R and used these receptor mutants to determine if tyrosine phosphorylation of IL27R is required to induce the transforming signal of JAK2-V617F. Upon initial cytokine removal, there is a slight lag in the growth of JAK2-V617F cells expressing an IL27R receptor that lacks both intracellular tyrosines, suggesting these tyrosines may play a role in signaling. However, this lag is transient and cytokine-independent growth becomes similar to cells expressing wildtype IL27R and JAK2-V617F. This suggests tyrosine phosphorylation of the receptor is not required to induce the transforming signal of JAK2-V617F. Our studies demonstrate for the first time that single chain components of heterodimeric receptors can functionally replace homodimeric receptors in mediating transformation of hematopoietic cells by JAK2-V617F. Our work also suggests that JAK2-V617F does not need to phosphorylate its receptor scaffold to mediate its transforming signal. The requirement for other endogenous cytokine receptors as substrates for activated JAK2-V617F receptors remains a possibility and is currently under investigation. Together, our studies demonstrate that IL27R, and perhaps other single chain components of heterodimeric receptors, may play unappreciated roles in myeloid diseases such as AML and MPNs.