Identifying Erlotinib As A Inhibitor Of Tyrosine Kinase JAK2～(V617F) And Defining Interactions Of Tyrosine Phosphatases SHP-1 With SHP-2

Reversible protein phosphorylation is a ubiquitous mechanism for the control of signal transduction networks that regulate diverse biological processes including cell development, proliferation, differentiation and apoptosis. For the key discovery of reversible protein phosphorylation, Edmond H. Fisher and Edwin G. Krebs were awarded the Nobel Prize for Physiology or Medicine in 1992. The phosphorylation state of a protein is controlled dynamically by both protein kinases and phosphatases. Protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), important part of protein kinases and phosphatases play a crucial role in normal cell development, and dysfunction of these enzymes has been implicated in human cancers.Polycythemia vera (PV) is a clonal hematologic disease characterized by hypersensitivity of hematopoietic progenitor cells to growth factors and cytokines. In March and April 2005, five groups including ours reported finding a gain-function mutation in the JAK homology 2 (JH2) domain of tyrosine kinase JAK2 that results in a valine to phenylalanine substitution at position 617 (V617F) associated with polycythemia vera and other related myeloproliferative disorders. The mutation is found in a significant percentage of patients with polycythemia vera (95%), idiopathic myelofibrosis (50%) and essential thrombocythemia (50%). Recent study interests were focused on the molecular pathogenesis in PV and the development of targeted inhibitor to treat the disease. The recent studies represented that the JAK2 mutant displays a much increased kinase activity and generates a PV-like phenotype in mouse bone marrow transplant models.In this study, we develop a JAK kinase assay in vitro. Then, the data of the JAK kinase assay shows that the anti-cancer drug Erlotinib (Tarceva) is a potent inhibitor of JAK2(V617F) activity. Furthermore, Similar inhibitory effects were found with the JAK2(V617F)-positive human erythroleukemia HEL cell line. These data suggest that Erlotinib may be used for treatment of JAK2(V617F)-positive PV and other myeloproliferative disorders as a potential drug.SHP-1 and SHP-2 are Src homology 2 (SH2) domain-containing tyrosine phosphatases. SHP-1 is highly expressed in hematopoietic cells and, at a lower level, in various nonhematopoietic cells, whereas SHP-2 is widely expressed. Both SHP-1 and SHP-2 have important pathological implications. In humans, reduction of SHP-1 gene expression is observed in natural killer cell lymphomas as well as other types of lymphomas/leukemias. Methylation of the SHP-1 promoter causes loss of SHP-1 expression in malignant T-lymphoma cells. On the other hand, activation mutation of SHP-2 causes Noonan syndrome. This gain-of-function mutation of SHP-2 is also associated with sporadic juvenile myelomonocytic leukemias (JMML), myelodysplasic syndrome(MS), acute lymphoblastic leukemia(ALL), and acute myelogenous leukemia(AML).SHP-1 and SHP-2 share significant overall sequence identity, but their biological functions are often opposite. SHP-1 is generally considered as a negative signal transducer and SHP-2 as a positive one. However, the precise role of each enzyme in shared signaling pathways is not well defined. In this study, we investigated the interaction of these two enzymes in a single cell system by knocking down their expressions with small interfering RNAs and analyzing the effects on epidermal growth factor signaling. Interestingly, knockdown of either SHP-1 or SHP-2 caused significant reduction in the activation of ERK1/2 but not AKT. The data revealed that both SHP-1 and SHP-2 have a positive role in epidermal growth factor-induced ERK1/2 activation that they act cooperatively rather than antagonistically. Furthermore, SHP-1, SHP-2, and Gab1 formed a signaling complex, and SHP-1 and SHP-2 interact with each other. The interaction of SHP-1 with Gab1 is mediated by SHP-2 because it was abrogated by knockdown of SHP-2, and SHP-2, but not SHP-1, binds directly to tyrosine-phosphorylated Gab1. The interaction of SHP-1 and SHP-2 may be responsible for previously unexpected novel regulatory mechanism of cell signaling by tyrosine phosphatases.