Hyperkinetic Jak2 tyrosine kinase signaling has been implicated in several human diseases including leukemia lymphoma myeloma and the myeloproliferative neoplasms. vimentin. It was present in DMSO treated cells but absent in G6 treated cells. HEL cells treated with G6 showed both time- and dose-dependent cleavage of vimentin as well as a marked reorganization of vimentin intermediate filaments within intact cells. In a mouse model of Jak2-V617F mediated human erythroleukemia G6 also decreased Nadifloxacin the levels of vimentin protein family of cytoplasmic tyrosine kinases. Other members of this family include Jak1 Jak3 and Tyk2 (1). Jak2 is activated by a variety of cytokines growth factors and G Protein-coupled Receptor (GPCR) ligands resulting in signaling Nadifloxacin cascades that regulate cell growth proliferation and death (1). Upon binding of the ligand to its specific receptor the receptor-associated Jak proteins are activated via a phosphorylation event. An activated Jak can in turn phosphorylate and activate the Signal Transducers and Activators of Transcription (STAT) family of transcription factors. Phosphorylated STATs dimerize and translocate to the nucleus where they modulate gene transcription (2 3 Thus the Jak/STAT pathway results in a signal cascade from binding and activation at the plasma membrane to changes in gene transcription in the nucleus. Hyperkinetic Nadifloxacin Jak2 promotes cell growth and prevents apoptosis. Hence constitutively active Jak/STAT signaling pathway has been implicated in a variety of neoplastic disorders. Jak2 can become constitutively active by several different gene alterations including specific chromosomal translocations and point mutations. Jak2 chromosomal translocations such as TEL-Jak2 REL-Jak2 BCR-Jak2 and PCM1-Jak2 lead to the development of a variety of leukemias lymphomas and myelomas (4–10). Additionally an activating Jak2 point mutation (Jak2-V617F) has been linked to the myeloproliferative neoplasms (MPN) such as polycythemia vera essential thrombocythemia and primary myelofibrosis (11–15). This valine to phenylalanine substitution mutation present in codon 617 of the autoinhibitory pseudokinase domain of Jak2 allows the kinase to evade negative regulation thereby making it constitutively active. MPN patients bear this mutation in their marrow derived stem cells and are characterized by the overproduction of terminally differentiated blood cells of the myeloid lineage such as red cells or platelets. Current therapies for MPN patients include phlebotomy and hydroxyurea. While these treatments alleviate some disease symptomologies they are not curative in any way. Therefore there is an unmet clinical need for these patients. Using structure-based virtual screening our group recently identified a novel stilbenoid small molecule inhibitor of Jak2 named G6 (16). We subsequently showed that G6 specifically inhibits Jak2 mediated human pathologic cell growth (17 18 We also demonstrated that G6 inhibits Jak2 mediated cell proliferation via the suppression of key signaling molecules of the Jak/STAT pathway; the consequence of this inhibition is G1/S cell cycle arrest and apoptosis (17 18 Here we sought to elucidate the molecular and biochemical mechanisms by which G6 inhibits Jak2-mediated cellular proliferation. For this we compared protein expression profiles between vehicle treated and G6 treated cells using two-dimensional gel electrophoresis. The intermediate filament protein Nadifloxacin vimentin was one protein that was differentially expressed between the two conditions. We therefore hypothesized that the mechanism by which G6 inhibits Jak2-dependent cell proliferation involves modification of this protein. In this study our data Nrp1 support this hypothesis as we show that G6-induced inhibition of Jak2-mediated pathogenic cell growth correlates with the specific cleavage and cellular reorganization of vimentin. EXPERIMENTAL PROCEDURES Drugs G6 obtained from the National Cancer Institute/Developmental Therapeutics Program (NCI/DTP) was solublized in dimethyl sulfoxide (DMSO) at a concentration of 10 mM and stored at ?20°C. Nadifloxacin Reagents AG490 Jak Inhibitor I PD98059 and PP2 were purchased from Calbiochem. Cycloheximide was purchased from Fisher Scientific. Caspase Inhibitor I (Z-VAD (OMe)-FMK) Calpain Inhibitor V (Mu-Val-HPh-CH2F Mu = morpholinoureidyl; HPh = homophenylalanyl) Verapamil BAPTA-AM {“type”:”entrez-nucleotide” attrs.