Interaction between Salt-inducible Kinase 2 (SIK2) and p97/Valosin-containing Protein (VCP) Regulates Endoplasmic Reticulum (ER)-associated Protein Degradation in Mammalian Cells

Salt-inducible kinase 2 (SIK2) is an important regulator of cAMP response element-binding protein-mediated gene expression in various cell types and is the only AMP-activated protein kinase family member known to interact with the p97/valosin-containing protein (VCP) ATPase. Previously, we have demonstrated that SIK2 can regulate autophagy when proteasomal function is compromised. Here we report that physical and functional interactions between SIK2 and p97/VCP underlie the regulation of endoplasmic reticulum (ER)-associated protein degradation (ERAD). SIK2 co-localizes with p97/VCP in the ER membrane and stimulates its ATPase activity through direct phosphorylation. Although the expression of wild-type recombinant SIK2 accelerated the degradation and removal of ERAD substrates, the kinase-deficient variant conversely had no effect. Furthermore, down-regulation of endogenous SIK2 or mutation of the SIK2 target site on p97/VCP led to impaired degradation of ERAD substrates and disruption of ER homeostasis. Collectively, these findings highlight a mechanism by which the interplay between SIK2 and p97/VCP contributes to the regulation of ERAD in mammalian cells.     

Suppressor of Cytokine Signaling 6 (SOCS6) Negatively Regulates Flt3 Signal Transduction through Direct Binding to Phosphorylated Tyrosines 591 and 919 of Flt3

The receptor tyrosine kinase Flt3 is an important growth factor receptor in hematopoiesis, and gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia. SOCS6 (suppressor of cytokine signaling 6) is a member of the SOCS family of E3 ubiquitin ligases that can regulate receptor tyrosine kinase signal transduction. In this study, we analyzed the role of SOCS6 in Flt3 signal transduction. The results show that ligand stimulation of Flt3 can induce association of SOCS6 and Flt3 and tyrosine phosphorylation of SOCS6. Phosphopeptide fishing indicated that SOCS6 binds directly to phosphotyrosines 591 and 919 of Flt3. By using stably transfected Ba/F3 cells with Flt3 and/or SOCS6, we show that the presence of SOCS6 can enhance ubiquitination of Flt3, as well as internalization and degradation of the receptor. The presence of SOCS6 also induces weaker activation of Erk1/2, but not Akt, in transfected Ba/F3 and UT-7 cells and in OCI-AML-5 cells. The absence of SOCS6 promotes Ba/F3 and UT-7 cell proliferation induced by oncogenic internal tandem duplications of Flt3. Taken together, these results suggest that SOCS6 negatively regulates Flt3 activation, the downstream Erk signaling pathway, and cell proliferation.     

Tissue-type Plasminogen Activator (tPA) Promotes M1 Macrophage Survival through p90 Ribosomal S6 Kinase (RSK) and p38 Mitogen-activated Protein Kinase (MAPK) Pathway

Macrophage accumulation is one of the hallmarks of progressive kidney disease. Resting macrophages have a finite lifespan, but become resistant to apoptosis in response to pathogenic cues, whereas the underlying mechanism remains unknown. Tissue-type plasminogen activator (tPA), a protease up-regulated in the kidneys with chronic injury, has been shown to promote macrophage accumulation and renal inflammation. We hypothesized that tPA may be the endogenous factor that promotes macrophage survival and extends their lifespan that leads to their accumulation in the injured kidneys. We examined the role of tPA in macrophage survival, and found that tPA protected macrophages from both staurosporine and H₂O₂-induced apoptosis. tPA promoted the survival of both resting and lipopolysaccharide- or interferon-γ-induced M1 macrophages, but failed to do so in the interleukin 4 (IL4)-induced M2 macrophages. In the kidneys with unilateral ureteral obstruction, there were significantly more apoptotic M1 macrophages in tPA-deficient mice than their wild-type counterparts, and obstruction-induced M1 macrophages accumulation and M1 chemokine expression were markedly reduced in these knock-out mice. The cytoprotective effect of tPA required its receptor, LDL receptor-related protein-1 (LRP-1). tPA induced the phosphorylation of Erk1/2, p90 ribosomal S6 kinase (RSK), and p38 in a temporal order. The tPA-mediated macrophage survival was eliminated by PD98059, BI-D1870, or sc68376, the specific inhibitors for Erk1/2, p90RSK, or p38, respectively. Thus, it is clear that tPA promoted M1 macrophage survival through its receptor LRP-1-mediated novel signaling cascade involving Erk1/2, p90RSK, and p38, which leads to the accumulation of these cells in the injured kidneys.     

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.     

AMP-activated Protein Kinase (AMPK) Negatively Regulates Nox4-dependent Activation of p53 and Epithelial Cell Apoptosis in Diabetes

Diabetes and high glucose (HG) increase the generation of NADPH oxidase-derived reactive oxygen species and induce apoptosis of glomerular epithelial cells (podocytes). Loss of podocytes contributes to albuminuria, a major risk factor for progression of kidney disease. Here, we show that HG inactivates AMP-activated protein kinase (AMPK), up-regulates Nox4, enhances NADPH oxidase activity, and induces podocyte apoptosis. Activation of AMPK blocked HG-induced expression of Nox4, NADPH oxidase activity, and apoptosis. We also identified the tumor suppressor protein p53 as a mediator of podocyte apoptosis in cells exposed to HG. Inactivation of AMPK by HG up-regulated the expression and phosphorylation of p53, and p53 acted downstream of Nox4. To investigate the mechanism of podocyte apoptosis in vivo, we used OVE26 mice, a model of type 1 diabetes. Glomeruli isolated from these mice showed decreased phosphorylation of AMPK and enhanced expression of Nox4 and p53. Pharmacologic activation of AMPK by 5-aminoimidazole-4-carboxamide-1-riboside in OVE26 mice attenuated Nox4 and p53 expression. Administration of 5-aminoimidazole-4-carboxamide-1-riboside also prevented renal hypertrophy, glomerular basement thickening, foot process effacement, and podocyte loss, resulting in marked reduction in albuminuria. Our results uncover a novel function of AMPK that integrates metabolic input to Nox4 and provide new insight for activation of p53 to induce podocyte apoptosis. The data indicate the potential therapeutic utility of AMPK activators to block Nox4 and reactive oxygen species generation and to reduce urinary albumin excretion in type 1 diabetes.     

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.     

Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing p38/c-Jun N-terminal Kinase (JNK) Mitogen-activated Protein Kinase (MAPK)-mediated G2/M Progression Delay and Cell Apoptosis

Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A₂ (TxA₂)/TxA₂ receptor (T prostanoid receptor (TP)) modulates the progression of some carcinomas; however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266 and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 as being involved in modulating cell proliferation. We further incubated MM cells with prostaglandin receptor antagonists or agonists and found that only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cell G₂/M phase delay accompanied by reducing cyclin B1/cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G₂/M arrest. We also found that the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing led to the MM cell apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA₂/TP promotes MM cell proliferation by reducing cell delay at G₂/M phase via elevating p38 MAPK/JNK-mediated cyclin B1/CDK1 expression and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy.