Insulin and insulin analogs stimulate proliferation of human mammary epithelial cells. We identified and analyzed the signaling pathways related to cell proliferation induced by regular insulin and by four insulin analogs presently approved for therapeutical use. Benign and malignant mammary cell lines showing different insulin receptor (IR) and IGF-I receptor (IGF-IR) expression patterns were studied. Cell proliferation was studied by crystal violet staining (BrdU-FACS analysis). Activation of insulin and IGF signaling pathways was studied by analysis of the phosphorylation status of IGF-IR and of key signaling proteins of the phosphoinositide 3-kinase (PI3K)/Akt and MAP kinase pathways, by the use of specific PI3K and MAP kinase inhibitors, and by silencing of IR and IGF-IR. Lantus stimulated the growth of MCF7 cells, which show high IGF-IR/IR ratio, significantly at 0.3 nmol/l, while regular insulin (Actrapid and bovine insulin) and other insulin analogs (Novorapid, Humalog, and Levemir) stimulated cell growth at 1.5-15 nmol/l concentrations. No difference between Lantus and the other insulin analogs was observed regarding growth stimulation of MCF10A cells showing low IGF-IR/IR ratio. Growth stimulation of MCF7 cells by Lantus was mainly due to strong activation of the IGF-IR and the MAP kinase pathway. Regular insulin and other insulin analogs tested activated mainly the IR and the PI3K/Akt pathway. We conclude that unlike regular insulin and other insulin analogs, Lantus strongly activates the IGF-IR and the MAP kinase pathway in MCF7 cells and is a strong mitogen for cells characterized by a high-IGF-IR/IR ratio.

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Pseudokinases lack conserved motifs typically required for kinase activity. Nearly half of pseudokinases bind ATP, but only few retain phosphotransfer activity, leaving the functional role of nucleotide binding in most cases unknown. Janus kinases (JAKs) are nonreceptor tyrosine kinases with a tandem pseudokinase-kinase domain configuration, where the pseudokinase domain (JAK homology 2, JH2) has important regulatory functions and harbors mutations underlying hematological and immunological diseases. JH2 of JAK1, JAK2, and TYK2 all bind ATP, but the significance of this is unclear. We characterize the role of nucleotide binding in normal and pathogenic JAK signaling using comprehensive structure-based mutagenesis. Disruption of JH2 ATP binding in wildtype JAK2 has only minor effects, and in the presence of type I cytokine receptors, the mutations do not affect JAK2 activation. However, JH2 mutants devoid of ATP binding ameliorate the hyperactivation of JAK2 V617F. Disrupting ATP binding in JH2 also inhibits the hyperactivity of other pathogenic JAK2 mutants, as well as of JAK1 V658F, and prevents induction of erythrocytosis in a JAK2 V617F myeloproliferative neoplasm mouse model. Molecular dynamic simulations and thermal-shift analysis indicate that ATP binding stabilizes JH2, with a pronounced effect on the C helix region, which plays a critical role in pathogenic activation of JAK2. Taken together, our results suggest that ATP binding to JH2 serves a structural role in JAKs, which is required for aberrant activity of pathogenic JAK mutants. The inhibitory effect of abrogating JH2 ATP binding in pathogenic JAK mutants may warrant novel therapeutic approaches.T he Janus kinases (JAK1-3, TYK2) are a family of nonreceptor tyrosine kinases with essential functions in the regulation of hematopoiesis, the immune system, and cellular metabolism. JAKs interact specifically with various cytokine receptors and couple cytokine binding to cytoplasmic signaling cascades, including the signal transducers and activators of transcription (STAT) pathway. JAKs consist of an N-terminal FERM domain, an SH2-like (Src homology 2) domain, a pseudokinase domain (JAK homology 2, JH2), and the C-terminal tyrosine kinase domain (JH1). JH2 mediates critical regulatory functions in JAKs and primarily serves to inhibit basal JH1 activity. Experimental deletion of JH2 increases JH1 activity in full-length JAK in the absence of stimulation (1-3), and in recombinant systems addition of JH2 suppresses JH1 activity (4-6). JH2 is, however, also required for ligand-induced activation of full-length JAKs in cell (1-3, 6, 7). The regulatory functions of JH2 are corroborated by the multitude of human disease mutations identified in the domain. The most common JAK2 mutation, V617F, leads to cytokine-independent signaling through the exclusively JAK2-dependent homotypic receptors for erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), and thrombopoietin (8). The V617F mutation is found in ∼95% of patients with polycythemia vera (PV) (9...

Major progress has been recently made in understanding the molecular pathogenesis of myeloproliferative neoplasms (MPN). Mutations in one of four genes-JAK2, MPL, CALR, and CSF3R-can be found in the vast majority of patients with MPN and represent driver mutations that can induce the MPN phenotype. Hyperactive JAK/STAT signaling appears to be the common denominator of MPN, even in patients with CALR mutations and the so-called "triple-negative" MPN, where the driver gene mutation is still unknown. Mutations in epigenetic regulators, transcription factors, and signaling components modify the course of the disease and can contribute to disease initiation and/or progression. The central role of JAK2 in MPN allowed development of small molecular inhibitors that are in clinical use and are active in almost all patients with MPN. Advances in understanding the mechanism of JAK2 activation open new perspectives of developing the next generation of inhibitors that will be selective for the mutated forms of JAK2.