Mitogen-activated Protein Kinase (MAPK) Hyperactivation and Enhanced NRAS Expression Drive Acquired Vemurafenib Resistance in V600E BRAF Melanoma Cells

Although targeting the V600E activating mutation in the BRAF gene, the most common genetic abnormality in melanoma, has shown clinical efficacy in melanoma patients, response is, invariably, short lived. To better understand mechanisms underlying this acquisition of resistance to BRAF-targeted therapy in previously responsive melanomas, we induced vemurafenib resistance in two V600E BRAF+ve melanoma cell lines, A375 and DM443, by serial in vitro vemurafenib exposure. The resulting approximately 10-fold more vemurafenib-resistant cell lines, A375rVem and D443rVem, had higher growth rates and showed differential collateral resistance to cisplatin, melphalan, and temozolomide. The acquisition of vemurafenib resistance was associated with significantly increased NRAS levels in A375rVem and D443rVem, increased activation of the prosurvival protein, AKT, and the MAPKs, ERK, JNK, and P38, which correlated with decreased levels of the MAPK inhibitor protein, GSTP1. Despite the increased NRAS, whole exome sequencing showed no NRAS gene mutations. Inhibition of all three MAPKs and siRNA-mediated NRAS suppression both reversed vemurafenib resistance significantly in A375rVem and DM443rVem. Together, the results indicate a mechanism of acquired vemurafenib resistance in V600E BRAF+ve melanoma cells that involves increased activation of all three human MAPKs and the PI3K pathway, as well as increased NRAS expression, which, contrary to previous reports, was not associated with mutations in the NRAS gene. The data highlight the complexity of the acquired vemurafenib resistance phenotype and the challenge of optimizing BRAF-targeted therapy in this disease. They also suggest that targeting the MAPKs and/or NRAS may provide a strategy to mitigate such resistance in V600E BRAF+ve melanoma.     

A Residue Quartet in the Extracellular Domain of the Prolactin Receptor Selectively Controls Mitogen-activated Protein Kinase Signaling

Cytokine receptors elicit several signaling pathways, but it is poorly understood how they select and discriminate between them. We have scrutinized the prolactin receptor as an archetype model of homodimeric cytokine receptors to address the role of the extracellular membrane proximal domain in signal transfer and pathway selection. Structure-guided manipulation of residues involved in the receptor dimerization interface identified one residue (position 170) that in cell-based assays profoundly altered pathway selectivity and species-specific bio-characteristics. Subsequent in vitro spectroscopic and nuclear magnetic resonance analyses revealed that this residue was part of a residue quartet responsible for specific local structural changes underlying these effects. This included alteration of a novel aromatic T-stack within the membrane proximal domain, which promoted selective signaling affecting primarily the MAPK (ERK1/2) pathway. Importantly, activation of the MAPK pathway correlated with in vitro stabilities of ternary ligand·receptor complexes, suggesting a threshold mean lifetime of the complex necessary to achieve maximal activation. No such dependence was observed for STAT5 signaling. Thus, this study establishes a residue quartet in the extracellular membrane proximal domain of homodimeric cytokine receptors as a key regulator of intracellular signaling discrimination.     

Cyclic AMP Signaling Reduces Sirtuin 6 Expression in Non-small Cell Lung Cancer Cells by Promoting Ubiquitin-Proteasomal Degradation via Inhibition of the Raf-MEK-ERK (Raf/Mitogen-activated Extracellular Signal-regulated Kinase/Extracellular Signal-regulated Kinase) Pathway

The cAMP signaling system regulates various cellular functions, including metabolism, gene expression, and death. Sirtuin 6 (SIRT6) removes acetyl groups from histones and regulates genomic stability and cell viability. We hypothesized that cAMP modulates SIRT6 activity to regulate apoptosis. Therefore, we examined the effects of cAMP signaling on SIRT6 expression and radiation-induced apoptosis in lung cancer cells. cAMP signaling in H1299 and A549 human non-small cell lung cancer cells was activated via the expression of constitutively active Gα_s plus treatment with prostaglandin E2 (PGE2), isoproterenol, or forskolin. The expression of sirtuins and signaling molecules were analyzed by Western blotting. Activation of cAMP signaling reduced SIRT6 protein expression in lung cancer cells. cAMP signaling increased the ubiquitination of SIRT6 protein and promoted its degradation. Treatment with MG132 and inhibiting PKA with H89 or with a dominant-negative PKA abolished the cAMP-mediated reduction in SIRT6 levels. Treatment with PGE2 inhibited c-Raf activation by increasing inhibitory phosphorylation at Ser-259 in a PKA-dependent manner, thereby inhibiting downstream MEK-ERK signaling. Inhibiting ERK with inhibitors or with dominant-negative ERKs reduced SIRT6 expression, whereas activation of ERK by constitutively active MEK abolished the SIRT6-depleting effects of PGE2. cAMP signaling also augmented radiation-induced apoptosis in lung cancer cells. This effect was abolished by exogenous expression of SIRT6. It is concluded that cAMP signaling reduces SIRT6 expression by promoting its ubiquitin-proteasome-dependent degradation, a process mediated by the PKA-dependent inhibition of the Raf-MEK-ERK pathway. Reduced SIRT6 expression mediates the augmentation of radiation-induced apoptosis by cAMP signaling in lung cancer cells.     

Inhibition of G-protein-coupled Receptor Kinase 2 (GRK2) Triggers the Growth-promoting Mitogen-activated Protein Kinase (MAPK) Pathway

Inhibition of G-protein-coupled receptor kinase 2 (GRK2) is an emerging treatment option for heart failure. Because GRK2 is also indispensable for growth and development, we analyzed the impact of GRK2 inhibition on cell growth and proliferation. Inhibition of GRK2 by the dominant-negative GRK2-K220R did not affect the proliferation of cultured cells. In contrast, upon xenograft transplantation of cells into immunodeficient mice, the dominant-negative GRK2-K220R or a GRK2-specific peptide inhibitor increased tumor mass. The enhanced tumor growth upon GRK2 inhibition was attributed to the growth-promoting MAPK pathway because dual inhibition of the GRK2 and RAF-MAPK axis by the Raf kinase inhibitor protein (RKIP) did not increase tumor mass. The MAPK cascade contributed to the cardioprotective profile of GRK2 inhibition by preventing cardiomyocyte death, whereas dual inhibition of RAF/MAPK and GRK2 by RKIP induced cardiomyocyte apoptosis, cardiac dysfunction, and signs of heart failure. Thus, cardioprotective signaling induced by GRK2 inhibition is overlapping with tumor growth promotion.     

GABA(A) Receptor Pi (GABRP) Stimulates Basal-like Breast Cancer Cell Migration through Activation of Extracellular-regulated Kinase 1/2 (ERK1/2)

Breast cancer is a heterogeneous disease comprised of distinct subtypes predictive of patient outcome. Tumors of the basal-like subtype have a poor prognosis due to inherent aggressiveness and the lack of targeted therapeutics. Basal-like tumors typically lack estrogen receptor-α, progesterone receptor and HER2/ERBB2, or in other words they are triple negative (TN). Continued evaluation of basal-like breast cancer (BLBC) biology is essential to identify novel therapeutic targets. Expression of the pi subunit of the GABA(A) receptor (GABRP) is associated with the BLBC/TN subtype, and herein, we reveal its expression also correlates with metastases to the brain and poorer patient outcome. GABRP expression in breast cancer cell lines also demonstrates a significant correlation with the basal-like subtype suggesting that GABRP functions in the initiation and/or progression of basal-like tumors. To address this postulate, we stably silenced GABRP in two BLBC cell lines, HCC1187 and HCC70 cells. Decreased GABRP reduces in vitro tumorigenic potential and migration concurrent with alterations in the cytoskeleton, specifically diminished cellular protrusions and expression of the BLBC-associated cytokeratins, KRT5, KRT6B, KRT14, and KRT17. Silencing GABRP also decreases phosphorylation of extracellular regulated kinase 1/2 (ERK1/2) in both cell lines and selective inhibition of ERK1/2 similarly decreases the basal-like cytokeratins as well as migration. Combined, these data reveal a GABRP-ERK1/2-cytokeratin axis that maintains the migratory phenotype of basal-like breast cancer. GABRP is a component of a cell surface receptor, thus, these findings suggest that targeting this new signaling axis may have therapeutic potential in BLBC.     

F-Box Only Protein 31 (FBXO31) Negatively Regulates p38 Mitogen-activated Protein Kinase (MAPK) Signaling by Mediating Lysine 48-linked Ubiquitination and Degradation of Mitogen-activated Protein Kinase Kinase 6 (MKK6)

The p38 MAPK signal transduction pathway plays an important role in inflammatory and stress responses. MAPKK6 (MKK6), a dual specificity protein kinase, is a p38 activator. Activation of the MKK6-p38 pathway is kept in check by multiple layers of regulations, including autoinhibition, dimerization, scaffold proteins, and Lys-63-linked polyubiquitination. However, the mechanisms underlying deactivation of MKK6-p38, which is crucial for maintaining the magnitude and duration of signal transduction, are not well understood. Lys-48-linked ubiquitination, which marks substrates for proteasomal degradation, is an important negative posttranslational regulatory machinery for signal pathway transduction. Here we report that the accumulation of F-box only protein 31 (FBXO31), a component of Skp1·Cul1·F-box protein E3 ligase, negatively regulated p38 activation in cancer cells upon genotoxic stresses. Our results show that FBXO31 binds to MKK6 and mediates its Lys-48-linked polyubiquitination and degradation, thereby functioning as a negative regulator of MKK6-p38 signaling and protecting cells from stress-induced cell apoptosis. Taken together, our findings uncover a new mechanism of deactivation of MKK6-p38 and substantiate a novel regulatory role of FBXO31 in stress response.     

Glucose Activates TORC2-Gad8 Protein via Positive Regulation of the cAMP/cAMP-dependent Protein Kinase A (PKA) Pathway and Negative Regulation of the Pmk1 Protein-Mitogen-activated Protein Kinase Pathway

The target of rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol 3-kinase-related kinases. TOR proteins are found at the core of two evolutionary conserved complexes, known as TORC1 and TORC2. In fission yeast, TORC2 is dispensable for proliferation under optimal growth conditions but is required for starvation and stress responses. TORC2 has been implicated in a wide variety of functions; however, the signals that regulate TORC2 activity have so far remained obscure. TORC2 has one known direct substrate, the AGC kinase Gad8, which is related to AKT in human cells. Gad8 is phosphorylated by TORC2 at Ser-546 (equivalent to AKT Ser-473), leading to its activation. Here, we show that glucose is necessary and sufficient to induce Gad8 Ser-546 phosphorylation in vivo and Gad8 kinase activity in vitro. The glucose signal that activates TORC2-Gad8 is mediated via the cAMP/PKA pathway, a major glucose-sensing pathway. By contrast, Pmk1, similar to human extracellular signal-regulated kinases and a major stress-induced mitogen activated protein kinase (MAPK) in fission yeast, inhibits TORC2-dependent Gad8 phosphorylation and activation. Inhibition of TORC2-Gad8 also occurs in response to ionic or osmotic stress, in a manner dependent on the cAMP/PKA and Pmk1-MAPK signaling pathways. Our findings highlight the significance of glucose availability in regulation of TORC2-Gad8 and indicate a novel link between the cAMP/PKA, Pmk1/MAPK, and TORC2-Gad8 signaling.     

Inhibition of Protein Phosphatase 2A (PP2A) Prevents Mcl-1 Protein Dephosphorylation at the Thr-163/Ser-159 Phosphodegron,Dramatically Reducing Expression in Mcl-1-amplified Lymphoma Cells

Abundant, sustained expression of prosurvival Mcl-1 is an important determinant of viability and drug resistance in cancer cells. The Mcl-1 protein contains PEST sequences (enriched in proline, glutamic acid, serine, and threonine) and is normally subject to rapid turnover via multiple different pathways. One of these pathways involves a phosphodegron in the PEST region, where Thr-163 phosphorylation primes for Ser-159 phosphorylation by glycogen synthase kinase-3. Turnover via this phosphodegron-targeted pathway is reduced in Mcl-1-overexpressing BL41-3 Burkitt lymphoma and other cancer cells; turnover is further slowed in the presence of phorbol ester-induced ERK activation, resulting in Mcl-1 stabilization and an exacerbation of chemoresistance. The present studies focused on Mcl-1 dephosphorylation, which was also found to profoundly influence turnover. Exposure of BL41-3 cells to an inhibitor of protein phosphatase 2A (PP2A), okadaic acid, resulted in a rapid increase in phosphorylation at Thr-163 and Ser-159, along with a precipitous decrease in Mcl-1 expression. The decline in Mcl-1 expression preceded the appearance of cell death markers and was not slowed in the presence of phorbol ester. Upon exposure to calyculin A, which also potently inhibits PP2A, versus tautomycin, which does not, only the former increased Thr-163/Ser-159 phosphorylation and decreased Mcl-1 expression. Mcl-1 co-immunoprecipitated with PP2A upon transfection into CHO cells, and PP2A/Aα knockdown recapitulated the increase in Mcl-1 phosphorylation and decrease in expression. In sum, inhibition of PP2A prevents Mcl-1 dephosphorylation and results in rapid loss of this prosurvival protein in chemoresistant cancer cells.     

Mitogen-activated protein kinase (MAPK) phosphatase 3-mediated cross-talk between MAPKs ERK2 and p38alpha

MAPK phosphatase 3 (MKP3) is highly specific for ERK1/2 inactivation via dephosphorylation of both phosphotyrosine and phosphothreonine critical for enzymatic activation. Here, we show that MKP3 is able to effectively dephosphorylate the phosphotyrosine, but not phosphothreonine, in the activation loop of p38α in vitro and in intact cells. The catalytic constant of the MKP3 reaction for p38α is comparable with that for ERK2. Remarkably, MKP3, ERK2, and phosphorylated p38α can form a stable ternary complex in solution, and the phosphatase activity of MKP3 toward p38α substrate is allosterically regulated by ERK2-MKP3 interaction. This suggests that MKP3 not only controls the activities of ERK2 and p38α but also mediates cross-talk between these two MAPK pathways. The crystal structure of bisphosphorylated p38α has been determined at 2.1 Å resolution. Comparisons between the phosphorylated MAPK structures reveal the molecular basis of MKP3 substrate specificity.     

Poxviral Protein A52 Stimulates p38 Mitogen-activated Protein Kinase (MAPK) Activation by Causing Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) Self-association Leading to Transforming Growth Factor β-activated Kinase 1 (TAK1) Recruitment

Vaccinia virus encodes a number of proteins that inhibit and manipulate innate immune signaling pathways that also have a role in virulence. These include A52, a protein shown to inhibit IL-1- and Toll-like receptor-stimulated NFκB activation, via interaction with interleukin-1 receptor-associated kinase 2 (IRAK2). Interestingly, A52 was also found to activate p38 MAPK and thus enhance Toll-like receptor-dependent IL-10 induction, which was TRAF6-dependent, but the manner in which A52 manipulates TRAF6 to stimulate p38 activation was unclear. Here, we show that A52 has a non-canonical TRAF6-binding motif that is essential for TRAF6 binding and p38 activation but dispensable for NFκB inhibition and IRAK2 interaction. Wild-type A52, but not a mutant defective in p38 activation and TRAF6 binding (F154A), caused TRAF6 oligomerization and subsequent TRAF6-TAK1 association. The crystal structure of A52 shows that it adopts a Bcl2-like fold and exists as a dimer in solution. Residue Met-65 was identified as being located in the A52 dimer interface, and consistent with that, A52-M65E was impaired in its ability to dimerize. A52-M65E although capable of interacting with TRAF6, was unable to cause either TRAF6 self-association, induce the TRAF6-TAK1 association, or activate p38 MAPK. The results suggest that an A52 dimer causes TRAF6 self-association, leading to TAK1 recruitment and p38 activation. This reveals a molecular mechanism whereby poxviruses manipulate TRAF6 to activate MAPKs (which can be proviral) without stimulating antiviral NFκB activation.     

Shock Wave Treatment Enhances Cell Proliferation and Improves Wound Healing by ATP Release-coupled Extracellular Signal-regulated Kinase (ERK) Activation

Shock wave treatment accelerates impaired wound healing in diverse clinical situations. However, the mechanisms underlying the beneficial effects of shock waves have not yet been fully revealed. Because cell proliferation is a major requirement in the wound healing cascade, we used in vitro studies and an in vivo wound healing model to study whether shock wave treatment influences proliferation by altering major extracellular factors and signaling pathways involved in cell proliferation. We identified extracellular ATP, released in an energy- and pulse number-dependent manner, as a trigger of the biological effects of shock wave treatment. Shock wave treatment induced ATP release, increased Erk1/2 and p38 MAPK activation, and enhanced proliferation in three different cell types (C3H10T1/2 murine mesenchymal progenitor cells, primary human adipose tissue-derived stem cells, and a human Jurkat T cell line) in vitro. Purinergic signaling-induced Erk1/2 activation was found to be essential for this proliferative effect, which was further confirmed by in vivo studies in a rat wound healing model where shock wave treatment induced proliferation and increased wound healing in an Erk1/2-dependent fashion. In summary, this report demonstrates that shock wave treatment triggers release of cellular ATP, which subsequently activates purinergic receptors and finally enhances proliferation in vitro and in vivo via downstream Erk1/2 signaling. In conclusion, our findings shed further light on the molecular mechanisms by which shock wave treatment exerts its beneficial effects. These findings could help to improve the clinical use of shock wave treatment for wound healing.