Diversity and versatility of p38 kinase signalling in health and disease.

The ability of cells to deal with different types of stressful situations in a precise and coordinated manner is key for survival and involves various signalling networks. Over the past 25 years, p38 kinases - in particular, p38α - have been implicated in the cellular response to stress at many levels. These span from environmental and intracellular stresses, such as hyperosmolarity, oxidative stress or DNA damage, to physiological situations that involve important cellular changes such as differentiation. Given that p38α controls a plethora of functions, dysregulation of this pathway has been linked to diseases such as inflammation, immune disorders or cancer, suggesting the possibility that targeting p38α could be of therapeutic interest. In this Review, we discuss the organization of this signalling pathway focusing on the diversity of p38α substrates, their mechanisms and their links to particular cellular functions. We then address how the different cellular responses can be generated depending on the signal received and the cell type, and highlight the roles of this kinase in human physiology and in pathological contexts.

Characterization of p38α Signaling Networks in Cancer Cells Using Quantitative Proteomics and Phosphoproteomics.

p38α (encoded by MAPK14) is a protein kinase that regulates cellular responses to almost all types of environmental and intracellular stresses. Upon activation, p38α phosphorylates many substrates both in the cytoplasm and nucleus, allowing this pathway to regulate a wide variety of cellular processes. While the role of p38α in the stress response has been widely investigated, its implication in cell homeostasis is less understood. To investigate the signaling networks regulated by p38α in proliferating cancer cells, we performed quantitative proteomic and phosphoproteomic analyses in breast cancer cells in which this pathway had been either genetically targeted or chemically inhibited. Our study identified with high confidence 35 proteins and 82 phosphoproteins (114 phosphosites) that are modulated by p38α and highlighted the implication of various protein kinases, including MK2 and mTOR, in the p38α-regulated signaling networks. Moreover, functional analyses revealed an important contribution of p38α to the regulation of cell adhesion, DNA replication, and RNA metabolism. Indeed, we provide experimental evidence supporting that p38α facilitates cancer cell adhesion and showed that this p38α function is likely mediated by the modulation of the adaptor protein ArgBP2. Collectively, our results illustrate the complexity of the p38α-regulated signaling networks, provide valuable information on p38α-dependent phosphorylation events in cancer cells, and document a mechanism by which p38α can regulate cell adhesion.

p38delta and PKD1: kinase switches for insulin secretion.

Insufficient production of the hormone insulin by pancreatic beta cells results in diabetes. In this issue, Sumara et al. (2009) report key roles for the protein kinases p38delta and PKD1 in the regulation of insulin secretion as well as in the survival of pancreatic beta cells.

TGFß drives immune evasion in genetically reconstituted colon cancer metastasis.

Most patients with colorectal cancer die as a result of the disease spreading to other organs. However, no prevalent mutations have been associated with metastatic colorectal cancers. Instead, particular features of the tumour microenvironment, such as lack of T-cell infiltration, low type 1 T-helper cell (TH1) activity and reduced immune cytotoxicity or increased TGFß levels predict adverse outcomes in patients with colorectal cancer. Here we analyse the interplay between genetic alterations and the tumour microenvironment by crossing mice bearing conditional alleles of four main colorectal cancer mutations in intestinal stem cells. Quadruple-mutant mice developed metastatic intestinal tumours that display key hallmarks of human microsatellite-stable colorectal cancers, including low mutational burden, T-cell exclusion and TGFß-activated stroma. Inhibition of the PD-1-PD-L1 immune checkpoint provoked a limited response in this model system. By contrast, inhibition of TGFß unleashed a potent and enduring cytotoxic T-cell response against tumour cells that prevented metastasis. In mice with progressive liver metastatic disease, blockade of TGFß signalling rendered tumours susceptible to anti-PD-1-PD-L1 therapy. Our data show that increased TGFß in the tumour microenvironment represents a primary mechanism of immune evasion that promotes T-cell exclusion and blocks acquisition of the TH1-effector phenotype. Immunotherapies directed against TGFß signalling may therefore have broad applications in treating patients with advanced colorectal cancer.

MK2 degradation as a sensor of signal intensity that controls stress-induced cell fate.

Cell survival in response to stress is determined by the coordination of various signaling pathways. The kinase p38α is activated by many stresses, but the intensity and duration of the signal depends on the stimuli. How different p38α-activation dynamics may impact cell life/death decisions is unclear. Here, we show that the p38α-signaling output in response to stress is modulated by the expression levels of the downstream kinase MK2. We demonstrate that p38α forms a complex with MK2 in nonstimulated mammalian cells. Upon pathway activation, p38α phosphorylates MK2, the complex dissociates, and MK2 is degraded. Interestingly, transient p38α activation allows MK2 reexpression, reassembly of the p38α-MK2 complex, and cell survival. In contrast, sustained p38α activation induced by severe stress interferes with p38α-MK2 interaction, resulting in irreversible MK2 loss and cell death. MK2 degradation is mediated by the E3 ubiquitin ligase MDM2, and we identify four lysine residues in MK2 that are directly ubiquitinated by MDM2. Expression of an MK2 mutant that cannot be ubiquitinated by MDM2 enhances the survival of stressed cells. Our results indicate that MK2 reexpression and binding to p38α is critical for cell viability in response to stress and illustrate how particular p38α-activation patterns induced by different signals shape the stress-induced cell fate.

Dual deletion of guanylyl cyclase-A and p38 mitogen-activated protein kinase in podocytes with aldosterone administration causes glomerular intra-capillary thrombi.

Natriuretic peptides exert not only blood-lowering but also kidney-protective effects through guanylyl cyclase-A (GC-A), a natriuretic peptide receptor. Signaling through GC-A has been shown to protect podocytes from aldosterone-induced glomerular injury, and a p38 mitogen-activated protein kinase (MAPK) inhibitor reduced glomerular injury in aldosterone-infused podocyte-specific GC-A knockout mice. To explore the role of p38 MAPK in podocytes, we constructed podocyte-specific p38 MAPK and GC-A double knockout mice (pod-double knockout mice). Unexpectedly, aldosterone-infused and high salt-fed (B-ALDO)-treated pod-double knockout mice resulted in elevated serum creatinine, massive albuminuria, macrophage infiltration, foot process effacement, nephrin and podocin reduction, and additionally, intra-capillary fibrin thrombi, indicating endothelial injury. Microarray analysis showed increased plasminogen activator inhibitor-1 (PAI-1) in glomeruli of B-ALDO-treated pod-double knockout mice. In B-ALDO-treated pod-double knockout mice, PAI-1 increased in podocytes, and treatment with PAI-1 neutralizing antibody ameliorated intra-capillary thrombus formation. In vitro, deletion of p38 MAPK by the CRISPR/Cas9 system and knockdown of GC-A in human cultured podocytes upregulated PAI-1 and transforming growth factor- ß1 (TGF-ß1). When p38 MAPK knockout podocytes, transfected with a small interfering RNA to suppress GC-A, were co-cultured with glomerular endothelial cells in a transwell system, the expression of TGF-ß1 was increased in glomerular endothelial cells. PAI-1 inhibition ameliorated both podocyte and endothelial injury in the transwell system signifying elevated PAI-1 in podocytes is a factor disrupting normal podocyte-endothelial crosstalk. Thus, our results indicate that genetic dual deletion of p38 MAPK and GC-A in podocytes accelerates both podocyte and endothelial injuries, suggesting these two molecules play indispensable roles in podocyte function.

Regulation of death receptor signaling by the autophagy protein TP53INP2.

TP53INP2 positively regulates autophagy by binding to Atg8 proteins. Here, we uncover a novel role of TP53INP2 in death-receptor signaling. TP53INP2 sensitizes cells to apoptosis induced by death receptor ligands. In keeping with this, TP53INP2 deficiency in cultured cells or mouse livers protects against death receptor-induced apoptosis. TP53INP2 binds caspase-8 and the ubiquitin ligase TRAF6, thereby promoting the ubiquitination and activation of caspase-8 by TRAF6. We have defined a TRAF6-interacting motif (TIM) and a ubiquitin-interacting motif in TP53INP2, enabling it to function as a scaffold bridging already ubiquitinated caspase-8 to TRAF6 for further polyubiquitination of caspase-8. Mutations of key TIM residues in TP53INP2 abrogate its interaction with TRAF6 and caspase-8, and subsequently reduce levels of death receptor-induced apoptosis. A screen of cancer cell lines showed that those with higher protein levels of TP53INP2 are more prone to TRAIL-induced apoptosis, making TP53INP2 a potential predictive marker of cancer cell responsiveness to TRAIL treatment. These findings uncover a novel mechanism for the regulation of caspase-8 ubiquitination and reveal TP53INP2 as an important regulator of the death receptor pathway.

Requirement for epithelial p38α in KRAS-driven lung tumor progression.

Malignant transformation entails important changes in the control of cell proliferation through the rewiring of selected signaling pathways. Cancer cells then become very dependent on the proper function of those pathways, and their inhibition offers therapeutic opportunities. Here we identify the stress kinase p38α as a nononcogenic signaling molecule that enables the progression of KrasG12V-driven lung cancer. We demonstrate in vivo that, despite acting as a tumor suppressor in healthy alveolar progenitor cells, p38α contributes to the proliferation and malignization of lung cancer epithelial cells. We show that high expression levels of p38α correlate with poor survival in lung adenocarcinoma patients, and that genetic or chemical inhibition of p38α halts tumor growth in lung cancer mouse models. Moreover, we reveal a lung cancer epithelial cell-autonomous function for p38α promoting the expression of TIMP-1, which in turn stimulates cell proliferation in an autocrine manner. Altogether, our results suggest that epithelial p38α promotes KrasG12V-driven lung cancer progression via maintenance of cellular self-growth stimulatory signals.

RINGO/Speedy proteins, a family of non-canonical activators of CDK1 and CDK2.

Cyclin-dependent kinases (CDKs) require the binding to a regulatory subunit to acquire enzymatic activity, and cyclins are the canonical CDK activators. However, there are specific situations in which CDKs can be activated by non-cyclin proteins that are less characterized. This review focuses on the family of RINGO/Speedy proteins, which have no sequence amino acid homology to cyclins but can bind to and activate CDK1 and CDK2. Interestingly, RINGO/Speedy proteins can activate CDKs under conditions in which CDK-cyclin complexes would not be active, and there is evidence that RINGO/Speedy-activated CDKs can phosphorylate different sites than the cyclin-activated CDKs. RINGO/Speedy proteins were originally described in Xenopus oocytes, but their roles in mammalian cells have also been addressed. We will summarize the properties of RINGO/Speedy proteins and how they trigger CDK activation, and discuss recent studies that characterized their physiological functions. In particular, studies using genetically modified mice have shown that RingoA, also known as Spy1, plays a key role in meiosis regulation. Emerging evidence also suggests a potential role for RingoA/Spy1 in cancer.

IκBα deficiency imposes a fetal phenotype to intestinal stem cells.

The intestinal epithelium is a paradigm of adult tissue in constant regeneration that is supported by intestinal stem cells (ISCs). The mechanisms regulating ISC homeostasis after injury are poorly understood. We previously demonstrated that IκBα, the main regulator of NF-κB, exerts alternative nuclear functions as cytokine sensor in a subset of PRC2-regulated genes. Here, we show that nuclear IκBα is present in the ISC compartment. Mice deficient for IκBα show altered intestinal cell differentiation with persistence of a fetal-like ISC phenotype, associated with aberrant PRC2 activity at specific loci. Moreover, IκBα-deficient intestinal cells produce morphologically aberrant organoids carrying a PRC2-dependent fetal-like transcriptional signature. DSS treatment, which induces acute damage in the colonic epithelium of mice, results in a temporary loss of nuclear P-IκBα and its subsequent accumulation in early CD44-positive regenerating areas. Importantly, IκBα-deficient mice show higher resistance to damage, likely due to the persistent fetal-like ISC phenotype. These results highlight intestinal IκBα as a chromatin sensor of inflammation in the ISC compartment.

p38α blocks brown adipose tissue thermogenesis through p38δ inhibition.

Adipose tissue has emerged as an important regulator of whole-body metabolism, and its capacity to dissipate energy in the form of heat has acquired a special relevance in recent years as potential treatment for obesity. In this context, the p38MAPK pathway has arisen as a key player in the thermogenic program because it is required for the activation of brown adipose tissue (BAT) thermogenesis and participates also in the transformation of white adipose tissue (WAT) into BAT-like depot called beige/brite tissue. Here, using mice that are deficient in p38α specifically in adipose tissue (p38αFab-KO), we unexpectedly found that lack of p38α protected against high-fat diet (HFD)-induced obesity. We also showed that p38αFab-KO mice presented higher energy expenditure due to increased BAT thermogenesis. Mechanistically, we found that lack of p38α resulted in the activation of the related protein kinase family member p38δ. Our results showed that p38δ is activated in BAT by cold exposure, and lack of this kinase specifically in adipose tissue (p38δ Fab-KO) resulted in overweight together with reduced energy expenditure and lower body and skin surface temperature in the BAT region. These observations indicate that p38α probably blocks BAT thermogenesis through p38δ inhibition. Consistent with the results obtained in animals, p38α was reduced in visceral and subcutaneous adipose tissue of subjects with obesity and was inversely correlated with body mass index (BMI). Altogether, we have elucidated a mechanism implicated in physiological BAT activation that has potential clinical implications for the treatment of obesity and related diseases such as diabetes.

Deconstructing ERK signaling in tumorigenesis.

Ras proteins play important protumorigenic roles by activating multiple signaling pathways. A study in this issue of Molecular Cell (Shin et al., 2010) dissects the implications of ERK2 signaling downstream of oncogenic Ras in the induction of epithelial-to-mesenchymal transition (EMT), cell motility, and invasion.

p38α regulates cytokine-induced IFNγ secretion via the Mnk1/eIF4E pathway in Th1 cells.

The p38 mitogen-activated protein kinase (MAPK) pathway is involved in the regulation of immune and inflammatory processes. We used p38α-conditional, p38ß-deficient and p38α/ß double-null mouse models to address the role of these two p38 MAPK in CD4+ T cells, and found that p38α deficiency causes these cells to hyperproliferate. Our studies indicate that both p38α and p38ß are dispensable for T helper cell type 1 (Th1) differentiation but, by controlling interferon (IFN)γ and tumor necrosis factor (TNF)α production, are critical for normal Th1 effector function. We found that both p38α and p38ß modulate T-cell receptor-induced IFNγ and TNFα production, whereas only p38α regulates cytokine-induced IFNγ production. The lack of p38α and p38ß did not affect transcription and mRNA stability of Ifng. However, the absence of p38α in Th1 cells resulted in a decreased MNK1 phosphorylation after cytokine activation, and MNK1 inhibition blocked IFNγ production. Our results indicate that p38α regulates IFNγ secretion through the activation of the MNK1/eIF4E pathway of translation initiation and identify specific functions for p38α and p38ß in T-cell proliferation.

The p57 CDKi integrates stress signals into cell-cycle progression to promote cell survival upon stress.

The p57(Kip2) cyclin-dependent kinase inhibitor (CDKi) has been implicated in embryogenesis, stem-cell senescence and pathologies, but little is known of its role in cell cycle control. Here, we show that p57(Kip2) is targeted by the p38 stress-activated protein kinase (SAPK). Phosphorylation of p57(Kip2) at T143 by p38 enhances its association with and inhibition of Cdk2, which results in cell-cycle delay upon stress. Genetic inactivation of the SAPK or the CDKi abolishes cell-cycle delay upon osmostress and results in decreased cell viability. Oxidative stress and ionomycin also induce p38-mediated phosphorylation of p57 and cells lacking p38 or p57 display reduced viability to these stresses. Therefore, cell survival to various stresses depends on p57 phosphorylation by p38 that inhibits CDK activity. Together, these findings provide a novel molecular mechanism by which cells can delay cell cycle progression to maximize cell survival upon stress.

Meiotic inactivation of Xenopus Myt1 by CDK/XRINGO, but not CDK/cyclin, via site-specific phosphorylation.

Cell-cycle progression is regulated by cyclin-dependent kinases (CDKs). CDK1 and CDK2 can be also activated by noncyclin proteins named RINGO/Speedy, which were identified as inducers of the G2/M transition in Xenopus oocytes. However, it is unclear how XRINGO triggers M phase entry in oocytes. We show here that XRINGO-activated CDKs can phosphorylate specific residues in the regulatory domain of Myt1, a Wee1 family kinase that plays a key role in the G2 arrest of oocytes. We have identified three Ser that are major phosphoacceptor sites for CDK/XRINGO but are poorly phosphorylated by CDK/cyclin. Phosphorylation of these Ser inhibits Myt1 activity, whereas their mutation makes Myt1 resistant to inhibition by CDK/XRINGO. Our results demonstrate that XRINGO-activated CDKs have different substrate specificity than the CDK/cyclin complexes. We also describe a mechanism of Myt1 regulation based on site-specific phosphorylation, which is likely to mediate the induction of G2/M transition in oocytes by XRINGO.

p38alpha MAP kinase is essential in lung stem and progenitor cell proliferation and differentiation.

Stem cell function is central for the maintenance of normal tissue homeostasis. Here we show that deletion of p38alpha mitogen-activated protein (MAP) kinase in adult mice results in increased proliferation and defective differentiation of lung stem and progenitor cells both in vivo and in vitro. We found that p38alpha positively regulates factors such as CCAAT/enhancer-binding protein that are required for lung cell differentiation. In addition, p38alpha controls self-renewal of the lung stem and progenitor cell population by inhibiting proliferation-inducing signals, most notably epidermal growth factor receptor. As a consequence, the inactivation of p38alpha leads to an immature and hyperproliferative lung epithelium that is highly sensitized to K-Ras(G12V)-induced tumorigenesis. Our results indicate that by coordinating proliferation and differentiation signals in lung stem and progenitor cells, p38alpha has a key role in the regulation of lung cell renewal and tumorigenesis.

New insights into RSK activation and hematopoietic cancer.

The tyrosine kinase receptor FGFR3 is thought to play a role in hematopoietic malignancies. A new study in this issue of Cancer Cell identifies the serine/threonine kinase RSK2 as a key substrate of FGFR3 in human t(4;14)-positive multiple myeloma (MM) cells. Constitutively active FGFR3 directly phosphorylates RSK2 on Tyr529, which primes RSK2 for activation by the kinases ERK1 and ERK2 (ERK1/2). In turn, RSK2 activity plays an important role in the survival of FGFR3-expressing MM cells.