Songwang Hou

p38γ MAPK Cooperates with c-Jun in trans-Activating Matrix Metalloproteinase 9

Mitogen-activated protein kinases (MAPKs) regulate gene expression through transcription factors. However, the precise mechanisms in this critical signal event are largely unknown. Here, we show that the transcription factor c-Jun is activated by p38γ MAPK, and the activated c-Jun then recruits p38γ as a cofactor into the matrix metalloproteinase 9 (MMP9) promoter to induce its trans-activation and cell invasion. This signaling event was initiated by hyperexpressed p38γ that led to increased c-Jun synthesis, MMP9 transcription, and MMP9-dependent invasion through p38γ interacting with c-Jun. p38γ requires phosphorylation and its C terminus to bind c-Jun, whereas both c-Jun and p38γ are required for the trans-activation of MMP9. The active p38γ/c-Jun/MMP9 pathway also exists in human colon cancer, and there is a coupling of increased p38γ and MMP9 expression in the primary tissues. These results reveal a new paradigm in which a MAPK acts both as an activator and a cofactor of a transcription factor to regulate gene expression leading to an invasive response.

PTPH1 Dephosphorylates and Cooperates with p38γ MAPK to Increase Ras Oncogenesis through PDZ-Mediated Interaction

Protein phosphatases are believed to coordinate with kinases to execute biological functions, but examples of such integrated activities, however, are still missing. In this report, we have identified protein tyrosine phosphatase H1 (PTPH1) as a specific phosphatase for p38gamma mitogen-activated protein kinase (MAPK) and shown their cooperative oncogenic activity through direct binding. p38gamma, a Ras effector known to act independent of its phosphorylation, was first shown to require its unique PDZ-binding motif to increase Ras transformation. Yeast two-hybrid screening and in vitro and in vivo analyses further identified PTPH1 as a specific p38gamma phosphatase through PDZ-mediated binding. Additional experiments showed that PTPH1 itself plays a role in Ras-dependent malignant growth in vitro and/or in mice by a mechanism depending on its p38gamma-binding activity. Moreover, Ras increases both p38gamma and PTPH1 protein expression and there is a coupling of increased p38gamma and PTPH1 protein expression in primary colon cancer tissues. These results reveal a coordinative oncogenic activity of a MAPK with its specific phosphatase and suggest that PDZ-mediated p38gamma/PTPH1 complex may be a novel target for Ras-dependent malignancies.

p38α Antagonizes p38γ Activity through c-Jun-dependent Ubiquitin-proteasome Pathways in Regulating Ras Transformation and Stress Response

p38 MAPK family consists of four isoform proteins (α, β, γ, and δ) that are activated by the same stimuli, but the information about how these proteins act together to yield a biological response is missing. Here we show a feed-forward mechanism by which p38α may regulate Ras transformation and stress response through depleting its family member p38γ protein via c-Jun-dependent ubiquitin-proteasome pathways. Analyses of MAPK kinase 6 (MKK6)-p38 fusion proteins showed that constitutively active p38α (MKK6-p38α) and p38γ (MKK6-p38γ) stimulates and inhibits c-Jun phosphorylation respectively, leading to a distinct AP-1 regulation. Depending on cell type and/or stimuli, p38α phosphorylation results in either Ras-transformation inhibition or a cell-death escalation that invariably couples with a decrease in p38γ protein expression. p38γ, on the other hand, increases Ras-dependent growth or inhibits stress induced cell-death independent of phosphorylation. In cells expressing both proteins, p38α phosphorylation decreases p38γ protein expression, whereas its inhibition increases cellular p38γ concentrations, indicating an active role of p38α phosphorylation in negatively regulating p38γ protein expression. Mechanistic analyses show that p38α requires c-Jun activation to deplete p38γ proteins by ubiquitin-proteasome pathways. These results suggest that p38α may, upon phosphorylation, act as a gatekeeper of the p38 MAPK family to yield a coordinative biological response through disrupting its antagonistic p38γ family protein.

Membrane phospholipid redistribution in cancer micro-particles and implications in the recruitment of cationic protein factors

Cancer cell-derived micro-particles (MPs) play important regulatory roles on cellular and system levels. These activities are attributed in part to protein factors carried by MPs. However, recruitment strategies for sequestering certain protein factors in MPs are poorly understood. In the current study, using exogenous and endogenously expressed phospholipid-binding probes, we investigated the distribution of membrane phospholipids in MPs as a potential mechanism for electrostatically enriching cationic protein factors in MPs. We detected a significant level of externalised phosphatidylethanolamine (PE) at the outer surface of MPs. This was accompanied, in the inner leaflet of the MP membrane, by a greater density of negatively charged phospholipids, particularly phosphatidylserine (PS). The local enrichment of PS in the inner surface of MPs was correlated with an elevated presence of small GTPases in a polybasic region (PBR)-dependent fashion. By employing a series of RhoA derivatives, including constitutively active and RhoA derivatives lacking a PBR, we could demonstrate that the congregation of RhoA in MPs was dependent on the presence of the PBR. A chimer with the fusion of PBR sequence alone to GFP significantly enhanced GFP localisation in MPs, indicative of a positive contribution of electrostatic interactions in RhoA recruitment to MPs. Using in silico thermodynamic simulations, we characterised the electrostatic interactions between PBR and anionic lipid membrane surface. In summary, the redistribution of membrane phospholipids in MPs has an impact on the local ionic density, and is likely a contributing factor in the electrostatic recruitment of membrane-associated proteins to MPs in a PBR-dependent fashion.

PTPH1 cooperates with vitamin D receptor to stimulate breast cancer growth through their mutual stabilization

Tyrosine phosphorylation is tightly regulated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), and has a critical role in malignant transformation and progression. Although PTKs have a well-established role in regulating breast cancer growth, contribution of PTPs remains mostly unknown. Here, we report that the tyrosine phosphatase PTPH1 stimulates breast cancer growth through regulating vitamin D receptor (VDR) expression. PTPH1 was shown to be overexpressed in 49% of primary breast cancer and levels of its protein expression positively correlate with the clinic metastasis, suggesting its oncogenic activity. Indeed, PTPH1 promotes breast cancer growth by a mechanism independent of its phosphatase activity, but dependent of its stimulatory effect on the nuclear receptor VDR protein expression and depletion of induced VDR abolishes the PTPH1 oncogenic activity. Additional analyses showed that PTPH1 binds VDR and increases its cytoplasmic accumulation, leading to their mutual stabilization and stable expression of a nuclear localization-deficient VDR abolishes the growth-inhibitory activity of the receptor independent of 1,25-dihydroxyvitamin D3. These results reveal a new paradigm in which a PTP may stimulate breast cancer growth through increasing cytoplasmic translocation of a nuclear receptor, leading to their mutual stabilization.

p38γ Mitogen-activated protein kinase signals through phosphorylating its phosphatase PTPH1 in regulating ras protein oncogenesis and stress response.

Background: A cross-talk between a kinase and phosphatase plays a critical role in determining cellular fate. Results: We report that p38γ phosphorylates its phosphatase PTPH1 in regulation of Ras oncogenesis and stress response. Conclusion: These results indicate that a MAPK can signal through its phosphatase. Significance: These studies reveal a novel mechanism by which a MAPK signals through its phosphatase to determine cellular outcomes. Phosphatase plays a crucial role in determining cellular fate by inactivating its substrate kinase, but it is not known whether a kinase can vice versa phosphorylate its phosphatase to execute this function. Protein-tyrosine phosphatase H1 (PTPH1) is a specific phosphatase of p38γ mitogen-activated protein kinase (MAPK) through PDZ binding, and here, we show that p38γ is also a PTPH1 kinase through which it executes its oncogenic activity and regulates stress response. PTPH1 was identified as a substrate of p38γ by unbiased proteomic analysis, and its resultant phosphorylation at Ser-459 occurs in vitro and in vivo through their complex formation. Genetic and pharmacological analyses showed further that Ser-459 phosphorylation is directly regulated by Ras signaling and is important for Ras, p38γ, and PTPH1 oncogenic activity. Moreover, experiments with physiological stimuli revealed a novel stress pathway from p38γ to PTPH1/Ser-459 phosphorylation in regulating cell growth and cell death by a mechanism dependent on cellular environments but independent of canonical MAPK activities. These results thus reveal a new mechanism by which a MAPK regulates Ras oncogenesis and stress response through directly phosphorylating its phosphatase.

Phosphorylation and Stabilization of Topoisomerase IIα Protein by p38γ Mitogen-activated Protein Kinase Sensitize Breast Cancer Cells to Its Poisons

Cancer drugs suppress tumor cell growth by inhibiting specific cellular targets. However, most drugs also activate several cellular nonspecific stress pathways, and the implications of these off-target effects are mostly unknown. Here, we report that p38γ, but not p38α, MAPK is specifically activated by treatment of breast cancer cells with topoisomerase II (Topo II) drugs, whereas paclitaxel (Taxol) does not have this effect. The activated p38γ in turn phosphorylates and stabilizes Topo IIα protein, and this enhances the growth inhibition by Topo II drugs. Moreover, p38γ activity was shown to be necessary and sufficient for Topo IIα expression, the drug-p38γ-Topo IIα axis is only detected in intrinsically sensitive but not resistant cells, and p38γ is co-overexpressed with Topo IIα protein in primary breast cancers. These results reveal a new paradigm in which p38γ actively regulates the drug-Topo IIα signal transduction, and this may be exploited to increase the therapeutic activity of Topo II drugs.

A One-Step Staining Probe for Phosphatidylethanolamine.

Phosphatidylethanolamine (PE) is an abundant phospholipid in cellular membranes, but relatively little is known about the kinetics of PE in biological membrane systems. Characterizing PE on a cellular level has been challenging owing to a lack of proper molecular tools. The lantibiotic duramycin and its structural analogue, cinnamycin, are currently the only known polypeptides that have an established stereospecific structure for binding membrane PE with high affinity and high specificity. These lantibiotics are recognized for their potential as molecular probes for studying PE kinetics in various membranes. However, owing to their antibiotic nature, duramycin and cinnamycin exhibit appreciable levels of cytotoxicity at low micromolar concentrations in cultured mammalian cells by inducing membrane distortion and possible PE redistribution. These issues can potentially complicate study design and data interpretation. Here, we report the construction of a molecular probe consisting of duramycin attached to the C terminus of green fluorescent protein (GFP) by a PEG linker at a stoichiometry of 1. The construct retained specific binding toward PE and essentially no cytotoxicity compared to native duramycin. The biological utilities of this probe were demonstrated in a number of cellular staining studies involving PE dynamics. The availability of a one‐step, nontoxic molecular probe for PE will enable characterization of the biology of this important phospholipid.

The K-Ras effector p38γ MAPK confers intrinsic resistance to tyrosine kinase inhibitors by stimulating EGFR transcription and EGFR dephosphorylation

Mutations in K-Ras and epidermal growth factor receptor (EGFR) are mutually exclusive, but it is not known how K-Ras activation inactivates EGFR, leading to resistance of cancer cells to anti-EGFR therapy. Here, we report that the K-Ras effector p38γ MAPK confers intrinsic resistance to small molecular tyrosine kinase inhibitors (TKIs) by concurrently stimulating EGFR gene transcription and protein dephosphorylation. We found that p38γ increases EGFR transcription by c-Jun-mediated promoter binding and stimulates EGFR dephosphorylation via activation of protein-tyrosine phosphatase H1 (PTPH1). Silencing the p38γ/c-Jun/PTPH1 signaling network increased sensitivities to TKIs in K-Ras mutant cells in which EGFR knockdown inhibited growth. Similar results were obtained with the p38γ-specific pharmacological inhibitor pirfenidone. These results indicate that in K-Ras mutant cancers, EGFR activity is regulated by the p38γ/c-Jun/PTPH1 signaling network, whose disruption may be a novel strategy to restore the sensitivity to TKIs.

Early endosome as a pathogenic target for antiphosphatidylethanolamine antibodies

Significance Autoimmunity against phosphatidylethanolamine (PE) was first reported more than three decades ago. Since then, a growing body of literature has documented the prevalence of aPE in a number of clinical conditions including thrombosis and repeated pregnancy loss, but the cellular target of anti-PE antibodies has remained elusive. The study presented here shows that the cellular vulnerability to PE binding is mediated by interactions that occur in the endosomal compartment, which lead to the activation of inflammatory mechanisms. Thus, our results provide knowledge about the pathogenic mechanism of aPE and have important implications for the diagnosis and treatment of this autoimmune condition. Phosphatidylethanolamine (PE) is a major phospholipid species with important roles in membrane trafficking and reorganization. Accumulating clinical data indicate that the presence of circulating antibodies against PE is positively correlated with the symptoms of antiphospholipid syndromes (APS), including thrombosis and repeated pregnancy loss. However, PE is generally sequestered inside a normal resting cell, and the mechanism by which circulating anti-PE antibodies access cellular PE remains unknown. The studies presented here were conducted with synthetic PE-binding agents, plasma samples from patients with anti-PE autoimmunity, and purified anti-PE antibodies. The results suggest that the cellular vulnerability to anti-PE antibodies may be mediated by the binding of PE molecules in the membrane of the early endosome. Endosomal PE binding led to functional changes in endothelial cells, including declines in proliferation and increases in the production of reactive oxygen species, as well as the expression of inflammatory molecules. Collectively, our findings provide insight into the etiology of anti-PE autoimmunity and, because endosomes are of central importance in almost all types of cells, could have important implications for a wide range of biological processes.