UCSF

Non-receptor protein tyrosine kinase Syk mediates signal transduction pathways downstream of immunoreceptors, integrins, and C-type lectins and is expressed in immune cells. Overexpression and aberrant activation of Syk is observed in numerous hematopoietic malignancies and autoimmunity. These studies examined whether constitutively active versions of Syk lead to deregulated signaling, aberrant function in hematopoietic cells, and generation of hematological malignancies. A previously characterized fusion protein TEL-Syk was analyzed for constitutive activity, hypersensitivity toward low-dose cytokine stimulation and generation of a myeproliferative neoplastic disease. TEL-Syk expressing fetal liver hematopoietic cells led to constitutive activation, induced myeloid expansion/dysmyelopoiesis and dyserythropoiesis, myelofibrosis, elevated circulating inflammatory cytokines and JAK2-independent phosphorylation of STAT5. Therefore TEL-Syk causes a pre-leukemic myeloid disorder rather than a lymphoid leukemia as previously published. We further examined whether Y(342, 346)A mutations within interdomain B of Syk lead to constitutive activation and deregulated signaling. Expression of Syk Y(342, 346)A in 293T cells led to constitutive phosphorylation of NTAL and other tyrosine containing targets, but demonstrated reduced autophosphorylation. Primary macrophages expressing Syk Y(342, 346)A phagocytosed IgG-opsonized SRBCs normally, but failed to mobilize calcium or transform Ba/F3 cells. Lastly, to address whether disruption in auto-regulatory motifs in Syk led to constitutive activation, we generated an allelic series within Syk, then determined the ability of these mutants to phosphorylate the downstream adaptor NTAL following co-expression in 293T cells. Although Syk mutants enhanced overall tyrosine phosphorylation, only Syk mutants W135A and Q149A increased autophosphorylation. Furthermore, DT-40 B-cells expressing these Syk mutants demonstrated a hypomorphic calcium mobilization response after BCR cross-linking as compared to cells expressing a wild-type Syk, suggesting that other regulatory mechanism may compensate for constitutive Syk activation. We also found that all Syk mutants did not transform Ba/F3 in the absence of IL-3 as compared to TEL-Syk. Our data demonstrates that TEL-Syk is a potent oncogene that drives a myeloproliferative neoplasm with robust myelofibrosis, and disruption of auto-regulatory motifs in Syk lead to constitutive NTAL and tyrosine phosphorylation. These studies provide a system to study deregulated Syk signaling in hematopoietic malignancies and additional evidence to target the kinase domain to treat human leukemias.