No abstract

Stat proteins (signal transducers and activators of transcription) are transcription factors which transmit signals from activated cytokine receptors to the nucleus. They are tyrosine phosphorylated by Jak kinases and dimerize through their SH2 domains (22,59). A functional domain structure of the Stat proteins has been established, and the regions responsible for dimerization, specific DNA binding, and transactivation have been identified (21,23,49,62). Stat5a, Stat5b, and Stat6 are the members of the Stat gene family (26) which have the highest amino acid homologies. These mammalian Stat factors are related to Marelle, a recently identified Stat protein important for Drosophila larval development (25, 76). The sequence homologies of Marelle with Stat5 and Stat6 are 37 and 34%, respectively. Stat5a and Stat5b are two highly related genes expressed in most tissues (3,31,41,51

Proteasomal proteolysis relies on the activity of six catalytically active proteasomal subunits (b1, b2, b5, b1i, b2i and b5i). Applying a functional proteomics approach, we used a recently developed activity-based, cell-permeable proteasome-specific probe that for the first time allows differential visualization of individual active proteasomal subunits in intact primary cells. In primary leukemia samples, we observed remarkable variability in the amounts of active b1/1i-, b2/2i-and b5/5i-type of subunits, contrasting with their constant protein expression. Bortezomib inhibited b5-and b1-type, but to a lesser extend b2-type of subunits in live primary cells in vitro and in vivo. When we adapted the bortezomib-sensitive human acute myeloid leukemia cell line HL-60 to bortezomib 40 nM (HL-60a), proteasomal activity profiling revealed an upregulation of active subunits, and residual b1/b5-type of activity could be visualized in the presence of bortezomib 20 nM, in contrast to control cells. In a panel of cell lines from hematologic malignancies, the ratio between b2-type and (b1 þ b5)-type of active proteasomal polypeptides mirrored different degrees of bortezomib sensitivity. We thus conclude that the proteasomal activity profile varies in primary leukemia cells, and that the pattern of proteasomal subunit activity influences the sensitivity of hematologic malignancies toward bortezomib. Leukemia (2007) 21, 84-92.

Inhibition of BCR-ABL

The possible role of homodimerization events in intracellular signal transduction triggered by the bipartite human interleukin-4 receptor was addressed. We generated cell lines functionally expressing derivatives of the two receptor subunits alpha and gamma, which allow for a specific and background-free experimental induction of intracellular homo- and heterodimers. A heterodimer of alpha and gamma released an intracellular signal, whereas a gamma-gamma homodimer did not. Unexpectedly, we found the intracellular domain of interleukin-4 receptor alpha chain to evoke cell proliferation and activation of tyrosine kinase Jak1 as well as of transcription factor Stat6 upon homodimerization. Both recruitment of the common gamma chain and activation of kinase Jak3 were shown to be dispensible for these processes.

Exposure of cells to protein tyrosine phosphatase (PTP) inhibitors causes an increase in the phosphotyrosine content of many cellular proteins. However, the level at which the primary signaling event is affected is still unclear. We show that Jaks are activated by tyrosine phosphorylation in cells that are brief ly exposed to the PTP inhibitor pervanadate (PV), resulting in tyrosine phosphorylation and functional activation of Stat6 (in addition to other Stats). Mutant cell lines that lack Jak1 activity fail to support PV-mediated Cytokines transmit their signals through transmembrane receptors that are physically associated with members of the Jak family of protein tyrosine kinases (PTK) (1-8). Aggregation of receptors, resulting in the association of Jaks in a receptor complex and a conformational change in the kinase domain, is an early step in cytokine receptor activation (2-11). Transphosphorylation of the conserved tyrosine residues in the kinase activation segments promotes kinase activity (11). The activated Jaks then phosphorylate the receptor-associated and downstream signaling molecules, including Stat proteins recruited to the receptor complex (1)(2)(3)(4)(5)(6)(7)(8)(12)(13)(14)(15).Protein tyrosine phosphatases (PTP) are associated with many cytokine receptors and are implicated in the downregulation of ligand-induced signaling through dephosphorylation of the activated Jaks and the receptors (15)(16)(17)(18)(19)(20). Dephosphorylation of activated Stat proteins is catalyzed by different PTP activities in the nucleus (21-23). We and others have found that cytokine receptor-associated PTP activities not only down-regulate cytokine-induced signals but also suppress the constitutive activities of Jak proteins (21,(24)(25)(26).

Interleukin-4 (IL-4) shows species-specific activity due to species-restricted interaction with the IL-4 receptor a (IL-4Ra) chain. The second subunit of a functional IL-4 receptor, the common 7 chain (yJ, is more promiscuous, since human IL-4 is able to activate IL-4 receptor complexes containing either human or murine common 7 receptor chain (yJ. We have stably transfected factor-dependent mouse cells of myeloid and lymphoid origin with combinations of human IL-4Ra and yc derivatives. In these cell lines, both human and murine yc receptors as well as IL-4Ra chains from both species are simultaneously expressed. Both versions of' yc readily form ternary complexes with either human IL-4 and human IL-4Ra or murine I t -4 and murine IL-4Ra. Due to distinct ligand-binding properties of human and murine yL, the two receptor complexes can be activated preferentially by different mutant variants of human IL-4. The contribution of murine common 7 chain to human TL-4-induced signal transduction is suppressed by an inhibitory antibody directed to the extracellular domain of the mouse yC. We present evidence that the two IL-4R complexes functionally interfere with each other and compete for responselimiting signalling components.

Interleukin (IL)-4,1 a pleiotropic modulator of the immune system (1), exerts its activity on target cells through the interleukin-4 receptor. Different cell types, including lymphoid and myeloid blood cells, express a bipartite IL-4 receptor (IL-4R), which consists of the IL-4R ␣-chain (IL-4R␣) (2) and the common ␥ receptor chain (␥c) (3). IL-4R␣ may also form a functional IL-4 receptor in conjunction with the IL-13 receptor instead of ␥c, especially in nonimmune cells (4). In vitro IL-4R␣ has also been shown to trigger intracellular signal transduction as a homodimer without participation of a heterologous receptor subunit (5, 6); however, the possible physiological relevance of IL-4R␣ homodimers is not yet known.IL-4-induced dimerization of receptor subunits results in the rapid onset of various cytoplasmic events. Janus kinases JAK1 and JAK3, which are associated with IL-4R␣ and ␥c, respectively (7, 8), become activated, probably by transphosphorylation, and as a result several other constituents of the activated receptor complex are phosphorylated. Tyrosine phosphorylation of IL-4R␣, IRS-2 (insulin receptor substrate 2, originally termed 4PS/IL-4-induced phosphorylation substrate), phosphatidylinositol 3-kinase, STAT6, and probably other proteins generate a network of protein-protein contacts mediated by interactions between phosphotyrosines and Src homology 2 domains (9). Despite structure-function studies by several laboratories (10 -16), it is only partially understood how these IL-4-induced molecular events lead to long term cellular processes such as suppression of apoptosis, proliferation, and differentiation.The aspect of IL-4R-triggered intracellular signal transduction that has been best characterized is the JAK-STAT pathway. STAT (signal transducer and activator of transcription) factors are central components of signaling cascades triggered by cytokine receptors and are phosphorylated in response to ligand stimulation through receptor-associated Janus kinases (JAKs). STAT factors then dimerize and translocate to the cell nucleus, where they interact with cognate DNA sequences of ␥-interferon-responsive sites (GASs) and modulate transcription of target genes (17).Following signal induction by the ligand, both the IL-4 receptor and the IL-13 receptor specifically mediate the activation of STAT6 (15, 18 -21). Gene regulation by STAT6 appears to be of central importance for IL-4-governed immune regulation, since STAT6 knockout mice are unable to develop Th2 cells; are impaired in cell surface expression of CD23, IL-4R␣, and major histocompatibility complex class II; and show a drastic defect in immunoglobulin class switching (22)(23)(24).Results obtained in the course of investigations on related cytokine receptors raised the question of whether STAT6 is the only STAT protein involved in IL-4-induced intracellular signaling. The IL-4 receptor shares the common ␥-subunit with the receptors for IL-2, , and all cytokine receptors utilizing the ␥c subunit, with the exception of the IL-4R, activate STAT5 ...