Dual-specificity phosphatase 5 controls the localized inhibition, propagation, and transforming potential of ERK signaling

Deregulated extracellular signal-regulated kinase (ERK) signaling drives cancer growth. Normally, ERK activity is self-limiting by the rapid inactivation of upstream kinases and delayed induction of dual-specificity MAP kinase phosphatases (MKPs/DUSPs). However, interactions between these feedback mechanisms are unclear. Here we show that, although the MKP DUSP5 both inactivates and anchors ERK in the nucleus, it paradoxically increases and prolongs cytoplasmic ERK activity. The latter effect is caused, at least in part, by the relief of ERK-mediated RAF inhibition. The importance of this spatiotemporal interaction between these distinct feedback mechanisms is illustrated by the fact that expression of oncogenic BRAF(V600E), a feedback-insensitive mutant RAF kinase, reprograms DUSP5 into a cell-wide ERK inhibitor that facilitates cell proliferation and transformation. In contrast, DUSP5 deletion causes BRAF(V600E)-induced ERK hyperactivation and cellular senescence. Thus, feedback interactions within the ERK pathway can regulate cell proliferation and transformation, and suggest oncogene-specific roles for DUSP5 in controlling ERK signaling and cell fate

Suppression of mutant Kirsten-RAS (KRASG12D)-driven pancreatic carcinogenesis by dual-specificity MAP kinase phosphatases 5 and 6

The cytoplasmic phosphatase DUSP6 and its nuclear counterpart DUSP5 are negative regulators of RAS/ERK signalling. Here we use deletion of either Dusp5 or Dusp6 to explore the roles of these phosphatases in a murine model of KRASG12D-driven pancreatic cancer. By 56-days, loss of either DUSP5 or DUSP6 causes a significant increase in KRASG12D-driven pancreatic hyperplasia. This is accompanied by increased pancreatic acinar to ductal metaplasia (ADM) and the development of pre-neoplastic pancreatic intraepithelial neoplasia (PanINs). In contrast, by 100-days, pancreatic hyperplasia is reversed with significant atrophy of pancreatic tissue and weight loss observed in animals lacking either DUSP5 or DUSP6. On further ageing, Dusp6−/− mice display accelerated development of metastatic pancreatic ductal adenocarcinoma (PDAC), while in Dusp5−/− animals, although PDAC development is increased this process is attenuated by atrophy of pancreatic acinar tissue and severe weight loss in some animals before cancer could progress. Our data suggest that despite a common target in the ERK MAP kinase, DUSP5 and DUSP6 play partially non-redundant roles in suppressing oncogenic KRASG12D signalling, thus retarding both tumour initiation and progression. Our data suggest that loss of either DUSP5 or DUSP6, as observed in certain human tumours, including the pancreas, could promote carcinogenesis

PTH1R Mutants Found in Patients with Primary Failure of Tooth Eruption Disrupt G-Protein Signaling

<div><p>Aim</p><p>Primary failure of tooth eruption (PFE) is causally linked to heterozygous mutations of the parathyroid hormone receptor (PTH1R) gene. The mutants described so far lead to exchange of amino acids or truncation of the protein that may result in structural changes of the expressed PTH1R. However, functional effects of these mutations have not been investigated yet.</p><p>Materials and Methods</p><p>In HEK293 cells, PTH1R wild type was co-transfected with selected PTH1R mutants identified in patients with PFE. The effects on activation of PTH-regulated intracellular signaling pathways were analyzed by ELISA and Western immunoblotting. Differential effects of wild type and mutated PTH1R on TRESK ion channel regulation were analyzed by electrophysiological recordings in <i>Xenopus laevis</i> oocytes.</p><p>Results</p><p>In HEK293 cells, activation of PTH1R wild type increases cAMP and in response activates cAMP-stimulated protein kinase as detected by phosphorylation of the vasodilator stimulated phosphoprotein (VASP). In contrast, the PTH1R mutants are functionally inactive and mutant PTH1R/Gly452Glu has a dominant negative effect on the signaling of PTH1R wild type. Confocal imaging revealed that wild type PTH1R is expressed on the cell surface, whereas PTH1R/Gly452Glu mutant is mostly retained inside the cell. Furthermore, in contrast to wild type PTH1R which substantially augmented K⁺ currents of TRESK channels, coupling of mutated PTH1R to TRESK channels was completely abolished.</p><p>Conclusions</p><p>PTH1R mutations affect intracellular PTH-regulated signaling <i>in vitro</i>. In patients with primary failure of tooth eruption defective signaling of PTH1R mutations is suggested to occur in dento-alveolar cells and thus may lead to impaired tooth movement.</p></div

Coexpression of PTH1 receptor and TRESK in Xenopus oocytes.

<p>(A) Application of 100 nM PTH augments TRESK current amplitude 2.62-fold (left trace). Current augmentation was abolished upon co-expression of receptor mutants PTH1R/Trp339stop (middle trace) or PTH1R/Gly452Glu (right trace), respectively. (B) Bar graphs display TRESK current amplitudes before (black) and after PTH application (grey) for wild type PTH1R and the two PTH1R mutants.</p

Membrane localization of PTH1R wild type and mutants.

<p>Membrane fractions of <i>Xenopus laevis</i> oocytes injected with cRNA of myc-tagged wild type (WT) and mutated PTH1R or H₂O were analyzed by Western immunoblotting. As revealed by myc-tag antibody specific signals (as indicated on the right) were detected in preparations of PTH1R wild type and PTH1R/Gly452Gly whereas these protein bands were absent in samples of PTH1R/Trp339stop and H₂O (negative control). Loading of equal amounts of protein is monitored by unspecific double bands (asterisk) in all samples. Quality and amount of cRNA injected into oocytes for heterologous expression of receptors is documented by RNA gel (lower panel).</p

Activation of PTH1R by PTH in PTH1R transfected HEK293 cells increases intracellular cAMP and induces PKA-triggered VASP phosphorylation.

<p>The upper panel depicts cAMP increase in HEK293 cells transfected with PTH1R (pcDNA3/PTH1R transfected) or with the vector only (pcDNA3 transfected). Cells were stimulated for up to 10 min with 10 or 100 nM PTH as indicated. For PTH-independent positive control cells were stimulated by 5 μM forskolin (as depicted). Data of thee independent experiments are presented as mean ± SEM, n = 3, p< 0.05 compared to the pcDNA3 control. Phosphorylation of VASP and loading control were documented by Western immunoblots with specific antibodies as indicated in the lower panel.</p

Stimulation and localization of PTH1R wild type or PTH1R mutants.

<p>(A) Extracts of stimulated (as indicated) HEK293 cells transfected with pcDNA3, pcDNA3:PTH1R wild type (WT), pcDNA3:PTH1R/Trp339stop or pcDNA3:PTH1R/Gly452Glu mutant were analyzed for VASP phosphorylation at the cAMP/PKA-preferred site Serine 157 (upper panel). Blotting against actin was used as loading control and used for quantification of VASP phosphorylation (lower panel). Representative blots of three independent experiments are shown. (B) Representative images from confocal imaging of YFP-tagged wild type (WT-YFP) and PTH1R/Gly452Glu (452Glu-YFP) in transfected HEK293 cells. CellMask deep red membrane dye was used for membrane staining. Scale bar 10 μM.</p