Trafficking, activation, and kinetics of delta F508-cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR were compared in stably transduced C127I mouse mammary epithelial cells. Western blots detected a small amount of fully glycosylated delta F508-CFTR Efflux of 125I was stimulated by forskolin with the same mean effective concentration (EC50; approximately 0.5 microM) for CFTR and delta F508-CFTR cells, but the maximum response was reduced more than fivefold and its latency increased approximately threefold in delta F508-CFTR cells. In delta F508-CFTR cells, 3-isobutyl-1-methylxanthine (IBMX; EC50 = 1.45 microM) and 8-cyclopentyl-1,3-dipropylxanthine (CPX; EC50 = 58 microM) increased the peak forskolin-stimulated efflux rate approximately 2.5-fold and decreased the time to peak. A sevenfold increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels accompanied potentiation of forskolin-induced 125I efflux by IBMX but not by CPX. Elevation of intracellular cAMP increased linear voltage-independent whole cell currents 30-fold in CFTR and 4-fold in delta F508-CFTR cells; the response rate in delta F508-CFTR cells was much slower. Single-channel currents were detected in 57 of 68 cell-attached patches from forskolin-prestimulated CFTR cells vs. 6 of 35 patches in delta F508-CFTR cells. Mean number of active channels per patch was 4.1 for CFTR [open probability (Po) = 0.34] and 0.2 for delta F508-CFTR (Po = 0.11). The lower Po of delta F508-CFTR resulted from an approximately threefold longer mean interburst interval. We estimate that forskolin-stimulated chloride conductance of delta F508-CFTR C127I cells is < 5% of CFTR cells. CPX is approximately 25-fold more potent than IBMX in potentiating delta F508-CFTR and may operate by a mechanism other than elevation of cAMP.

The signaling lipid phosphatidylinositol (3,4,5)-trisphosphate (PIP3) is a key regulator of cell proliferation, survival, and migration and the enzyme that dephosphorylates it, phosphatase and tensin homolog (PTEN), is an important tumor suppressor. As excess PIP3 signaling is a hallmark of many cancers, its suppression through activation of PTEN is a potential cancer intervention. Using a heterologous expression system in which human PTEN-GFP is expressed in Dictyostelium cells, we identified mutations in the membrane-binding regulatory interface that increase the recruitment of PTEN to the plasma membrane due to enhanced association with PI(4,5)P2. We engineered these into an enhanced PTEN (ePTEN) with approximately eightfold increased ability to suppress PIP3 signaling. Upon expression in human cells, ePTEN decreases PIP3 levels in the plasma membrane; phosphorylation of AKT, a major downstream event in PIP3 signaling; and cell proliferation and migration. Thus, the activation of PTEN can readjust PIP3 signaling and may serve as a feasible target for anticancer therapies.protein engineering | PI3 kinase signaling | membrane localization | chemotaxis | protein interaction

No abstract

PTEN is a PIP3 phosphatase that antagonizes oncogenic PI3-kinase signalling. Due to its critical role in suppressing the potent signalling pathway, it is one of the most mutated tumour suppressors, especially in brain tumours. It is generally thought that PTEN deficiencies predominantly result from either loss of expression or enzymatic activity. By analysing PTEN in malignant glioblastoma primary cells derived from 16 of our patients, we report mutations that block localization of PTEN at the plasma membrane and nucleus without affecting lipid phosphatase activity. Cellular and biochemical analyses as well as structural modelling revealed that two mutations disrupt intramolecular interaction of PTEN and open its conformation, enhancing polyubiquitination of PTEN and decreasing protein stability. Moreover, promoting mono-ubiquitination increases protein stability and nuclear localization of mutant PTEN. Thus, our findings provide a molecular mechanism for cancer-associated PTEN defects and may lead to a brain cancer treatment that targets PTEN mono-ubiquitination.