A6 model renal epithelial cells were stably transfected with enhanced green fluorescent protein (EGFP)-tagged alpha- or beta-subunits of the epithelial Na(+) channel (ENaC). Transfected RNA and proteins were both expressed in low abundance, similar to the endogenous levels of ENaC in native cells. In living cells, laser scanning confocal microscopy revealed a predominantly subapical distribution of EGFP-labeled subunits, suggesting a readily accessible pool of subunits available to participate in Na(+) transport. The basal level of Na(+) transport in the clonal lines was enhanced two- to fourfold relative to the parent line. Natriferic responses to insulin or aldosterone were similar in magnitude to the parent line, while forskolin-stimulated Na(+) transport was 64% greater than control in both the alpha- and beta-transfected lines. In response to forskolin, EGFP-labeled channel subunits traffic to the apical membrane. These data suggest that channel regulators, not the channel per se, form the rate-limiting step in response to insulin or aldosterone stimulation, while the number of channel subunits is important for basal as well as cAMP-stimulated Na(+) transport.
Bcl-2 expression is high in CLL and this appears to participate in disease progression. Zap70 expression is a poor prognostic marker for the disease whose function may be to contribute in a pathway promoting heightened bcl-2 levels. We recently identified a pathway to heightened bcl-2 transcriptional expression involving the bcl-2 promoter CRE (TGACGTCA) element and c-jun/ATF-2 downstream of JNK1. Indeed, JNK activation is a known survival pathway in B cell lymphoma (Gururajan, et al. Blood106: 1382, 2005). We confirmed a strict correspondence of Zap70 expression with JNK activity in CLL cell lysates by immunoblot identification of Zap70 and of phospho-S73-c-jun; and we found bcl-2 levels to be high in cells with Zap70 and JNK activity. We uncovered an adaptor/scaffolding pathway physically linking these mediators. By immunoprecipitation from lysates in a series of Zap70+ CLL’s we found JNK1 to coimmunoprecipitate with CrkL, a JNK1 scaffolding that binds by its SH3 domain to polyproline sequences of JNK1, and with p85 and Cbl and Zap70 as well as Syk. By contrast, using GST-p85 as bait in pull-down experiments, a selective interaction was found for Zap70 with p85, particularly of the p85 C-terminal SH2 domain, in lysates with heavy Y319 phosphorylation of Zap70. In parallel pull-downs with such lysates, exclusion of Cbl N-terminal TKB domain binding from Zap70 but not Syk was observed. Zap70+ CLL cell growth stimulated by the cytokine April was inhibited by the novel JNK inhibitor, CC-401, 5 uM, without significant effect on inhibition of NFkB or AKT activations. Such inhibition of JNK1 also led to upregulation of bim mRNA and downregulation of bcl-2 mRNA, and modest-to-moderate bcl-2 protein downregulation, in cells whose nuclear proteins contained abundant phospho-ATF2/c-jun binding activity for the bcl-2 promoter CRE site indicative of a transcriptional connection. Thus, Zap70 represents a point of origin in CLL cells for parallel pathways of Zap70 and Syk signaling to JNK1, involving specific complexes of Zap70 and Syk with p85/CrkL/JNK1(Cbl), respectively. These dual routes to JNK activation promote cell survival, in part, through the ability of JNK to contribute in a bcl-2 regulatory mechanism.
Here we demonstrate by immunoprecipitation and immunoblot, cbl is among the most heavily tyrosine phosphorylated adaptor proteins in primary AML blasts with Flt3 signaling, in the context of either mutation or overexpression/autocrine mechanisms. The human leukemic cell lines MV-4-11 and THP-1 model primary AML blasts in terms of Flt3 signaling by these respective criteria and demonstrate identical coupling between Flt3 and p85, the PI-3-kinase adaptor, by coimmunoprecipitation/blot experiments. Although cbl has no direct binding site on Flt3, it binds tightly to p85 SH2 by virtue of its tyrosine phosphorylation, also demonstrated by co-IP in cell lines and primary cells. Tyrosine phosphorylated cbl is a docking site for CrkII/L SH2’s and this provides a branch point for signals from Flt3 to PI-3-kinase or JNK, respectively, because CrkII(L) is known to bind JNK1 through SH3: polyproline interaction to serve as scaffolding; and interaction of JNK1 and CrkII/L was also observed by co-IP. In a survey of primary AML cases (n=33) there was a strict relationship between expression levels of (active) Flt3 and phospho-c-jun as readout for JNK activity level (p=0.001, r=0.54). To demonstrate the functional relevance of these interactions, siRNA knockdown of components was pursued in the cell lines and in primary AML blasts. JNK1 knockdown, and, to a much lesser degree, JNK2 knockdown, led to loss of phospho-c-jun expression in MV-4-11 and THP-1. Indeed, Flt3 signaling is required for JNK signaling because knockdown of Flt3 led to total loss of p-jun and c-jun expression in MV-4-11 and patient blast. By contrast, cbl knockdown led to selective loss of JNK signaling to p-jun without significantly affecting Flt3 or its downstream activating phosphorylation of AKT. Thus, despite binding by cbl to p85, cbl is not required for PI-3-kinase signaling because of redundancy supplied by the p85-Flt3 interaction. Further, by use of LY294002 to inhibit PI-3-kinase, PI-3-kinase is also not required for JNK signaling. However, selective inhibition of JNK signaling by the small molecule approach in these cell lines and in primary AML blasts leads to loss of proliferation, induction of apoptosis, and synergistic killing with daunorubicin. These observations form the platform for a phase I trial of JNK inhibition in refractory, multidrug-resistant, and Flt3-driven AML.
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