140,418 research outputs found
The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice
Nearly every extracellular ligand that has been found to play a role in regulating bone biology acts, at least in part, through MAPK pathways. Nevertheless, much remains to be learned about the contribution of MAPKs to osteoblast biology in vivo. Here we report that the p38 MAPK pathway is required for normal skeletogenesis in mice, as mice with deletion of any of the MAPK pathway member–encoding genes MAPK kinase 3 (Mkk3), Mkk6, p38a, or p38b displayed profoundly reduced bone mass secondary to defective osteoblast differentiation. Among the MAPK kinase kinase (MAP3K) family, we identified TGF-β–activated kinase 1 (TAK1; also known as MAP3K7) as the critical activator upstream of p38 in osteoblasts. Osteoblast-specific deletion of Tak1 resulted in clavicular hypoplasia and delayed fontanelle fusion, a phenotype similar to the cleidocranial dysplasia observed in humans haploinsufficient for the transcription factor runt-related transcription factor 2 (Runx2). Mechanistic analysis revealed that the TAK1–MKK3/6–p38 MAPK axis phosphorylated Runx2, promoting its association with the coactivator CREB-binding protein (CBP), which was required to regulate osteoblast genetic programs. These findings reveal an in vivo function for p38β and establish that MAPK signaling is essential for bone formation in vivo. These results also suggest that selective p38β agonists may represent attractive therapeutic agents to prevent bone loss associated with osteoporosis and aging
CXCL12-induced neurotoxicity critically depends on NMDA receptor-gated and L-type Ca2+ channels upstream of p38 MAPK.
BackgroundThe chemokine receptor CXCR4 (CD184) and its natural ligand CXCL12 contribute to many physiological processes, including decisions about cell death and survival in the central nervous system. In addition, CXCR4 is a co-receptor for human immunodeficiency virus (HIV)-1 and mediates the neurotoxicity of the viral envelope protein gp120. However, we previously observed that CXCL12 also causes toxicity in cerebrocortical neurons but the cellular mechanism remained incompletely defined.MethodsPrimary neuronal-glial cerebrocortical cell cultures from rat were exposed to a neurotoxicity-inducing CXCL12 concentration for different times and the activity of the stress-associated mitogen-activated protein kinase p38 (p38 MAPK) was assessed using an in vitro kinase assay. Neurotoxicity of CXCL12 and cellular localization of p38 MAPK was analyzed by immunofluorescence microscopy. Pharmacological inhibition of NMDA-type glutamate receptor-gated ion channels (NMDAR) of L-type Ca2+ channels was employed during 12- and 24-h exposure to neurotoxic amounts of CXCL12 to study the effects on active p38 MAPK and neuronal survival by Western blotting and microscopy, respectively. Neurotoxicity of CXCL12 was also assessed during pharmacological inhibition of p38 MAPK.ResultsHere, we show that a neurotoxic amount of CXCL12 triggers a significant increase of endogenous p38 MAPK activity in cerebrocortical cells. Immunofluorescence and Western blotting experiments with mixed neuronal-glial and neuron-depleted glial cerebrocortical cells revealed that the majority of active/phosphorylated p38 MAPK was located in neurons. Blockade of NMDAR-gated ion channels or L-type Ca2+ channels both abrogated an increase of active p38 MAPK and toxicity of CXCL12 in cerebrocortical neurons. Inhibition of L-type Ca2+ channels with nimodipine kept the active kinase at levels not significantly different from baseline while blocking NMDAR with MK-801 strongly reduced phosphorylated p38 MAPK below baseline. Finally, we confirmed that directly blocking p38 MAPK also abrogated neurotoxicity of CXCL12.ConclusionsOur findings link CXCL12-induced neuronal death to the regulation of NMDAR-gated ion channels and L-type Ca2+ channels upstream of p38 MAPK activation
Concurrent MEK targeted therapy prevents MAPK pathway reactivation during BRAFV600E targeted inhibition in a novel syngeneic murine glioma model.
Inhibitors of BRAFV600E kinase are currently under investigations in preclinical and clinical studies involving BRAFV600E glioma. Studies demonstrated clinical response to such individualized therapy in the majority of patients whereas in some patients tumors continue to grow despite treatment. To study resistance mechanisms, which include feedback activation of mitogen-activated protein kinase (MAPK) signaling in melanoma, we developed a luciferase-modified cell line (2341luc) from a BrafV600E mutant and Cdkn2a- deficient murine high-grade glioma, and analyzed its molecular responses to BRAFV600E- and MAPK kinase (MEK)-targeted inhibition. Immunocompetent, syngeneic FVB/N mice with intracranial grafts of 2341luc were tested for effects of BRAFV600E and MEK inhibitor treatments, with bioluminescence imaging up to 14-days after start of treatment and survival analysis as primary indicators of inhibitor activity. Intracranial injected tumor cells consistently generated high-grade glioma-like tumors in syngeneic mice. Intraperitoneal daily delivery of BRAFV600E inhibitor dabrafenib only transiently suppressed MAPK signaling, and rather increased Akt signaling and failed to extend survival for mice with intracranial 2341luc tumor. MEK inhibitor trametinib delivered by oral gavage daily suppressed MAPK pathway more effectively and had a more durable anti-growth effect than dabrafenib as well as a significant survival benefit. Compared with either agent alone, combined BRAFV600E and MEK inhibitor treatment was more effective in reducing tumor growth and extending animal subject survival, as corresponding to sustained MAPK pathway inhibition. Results derived from the 2341luc engraftment model application have clinical implications for the management of BRAFV600E glioma
FAK acts as a suppressor of RTK-MAP kinase signalling in Drosophila melanogaster epithelia and human cancer cells
Receptor Tyrosine Kinases (RTKs) and Focal Adhesion Kinase (FAK) regulate multiple signalling pathways, including mitogen-activated protein (MAP) kinase pathway. FAK interacts with several RTKs but little is known about how FAK regulates their downstream signalling. Here we investigated how FAK regulates signalling resulting from the overexpression of the RTKs RET and EGFR. FAK suppressed RTKs signalling in Drosophila melanogaster epithelia by impairing MAPK pathway. This regulation was also observed in MDA-MB-231 human breast cancer cells, suggesting it is a conserved phenomenon in humans. Mechanistically, FAK reduced receptor recycling into the plasma membrane, which resulted in lower MAPK activation. Conversely, increasing the membrane pool of the receptor increased MAPK pathway signalling. FAK is widely considered as a therapeutic target in cancer biology; however, it also has tumour suppressor properties in some contexts. Therefore, the FAK-mediated negative regulation of RTK/MAPK signalling described here may have potential implications in the designing of therapy strategies for RTK-driven tumours
The role of phosphatidylinositide-3-kinase in basal mitogen-activated protein kinase activity and cell survival
AbstractPhosphatidylinositide-3-OH-kinase (PI 3-kinase) is an upstream activator of p42/p44 mitogen-activated protein kinase (MAPK), but the role of PI 3-kinase-dependent MAPK remains obscure. Here we demonstrate that in a variety of different cell types, PI 3-kinase inhibition results in an inhibition of MAPK in unstimulated cells but does not interfere with growth factor-, or TPA-induced MAPK activity. Furthermore, inhibition of either PI 3-kinase or MEK/MAPK results in cell death in serum-starved cells. We concluded that basal, but not induced MAPK activity is mediated by PI 3-kinase and that this PI 3-kinase-mediated MEK/MAPK activity is essential for cell survival in quiescent cells
A human MAP kinase interactome.
Mitogen-activated protein kinase (MAPK) pathways form the backbone of signal transduction in the mammalian cell. Here we applied a systematic experimental and computational approach to map 2,269 interactions between human MAPK-related proteins and other cellular machinery and to assemble these data into functional modules. Multiple lines of evidence including conservation with yeast supported a core network of 641 interactions. Using small interfering RNA knockdowns, we observed that approximately one-third of MAPK-interacting proteins modulated MAPK-mediated signaling. We uncovered the Na-H exchanger NHE1 as a potential MAPK scaffold, found links between HSP90 chaperones and MAPK pathways and identified MUC12 as the human analog to the yeast signaling mucin Msb2. This study makes available a large resource of MAPK interactions and clone libraries, and it illustrates a methodology for probing signaling networks based on functional refinement of experimentally derived protein-interaction maps
Structural Mechanism for the Specific Assembly and Activation of the Extracellular Signal Regulated Kinase 5 (ERK5) Module
Mitogen-activated protein kinase (MAPK) activation depends on a linear binding motif found in all MAPK kinases (MKK). In addition, the PB1 (Phox and Bem1) domain of MKK5 is required for extracellular signal regulated kinase 5 (ERK5) activation. We present the crystal structure of ERK5 in complex with an MKK5 construct comprised of the PB1 domain and the linear binding motif. We show that ERK5 has distinct protein-protein interaction surfaces compared with ERK2, which is the closest ERK5 paralog. The two MAPKs have characteristically different physiological functions and their distinct protein-protein interaction surface topography enables them to bind different sets of activators and substrates. Structural and biochemical characterization revealed that the MKK5 PB1 domain cooperates with the MAPK binding linear motif to achieve substrate specific binding, and it also enables co-recruitment of the upstream activating enzyme and the downstream substrate into one signaling competent complex. Studies on present day MAPKs and MKKs hint on the way protein kinase networks may evolve. In particular, they suggest how paralogous enzymes with similar catalytic properties could acquire novel signaling roles by merely changing the way they make physical links to other proteins
Regulation and Function of Stress-Activated Protein Kinase Signal Transduction Pathways: A Dissertation
The c-Jun NH2-terminal kinase (JNK) group and the p38 group of mitogen-activated protein kinases (MAPK) are stress-activated protein kinases that regulate cell proliferation, differentiation, development, and apoptosis. These protein kinases are involved in a signal transduction cascade that includes a MAP kinase (MAPK), a MAP kinase kinase (MAP2K), and a MAP kinase kinase kinase (MAP3K). MAPK are phosphorylated and activated by the MAP2K, which are phosphorylated and activated by various MAP3K.
The work presented in this dissertation focuses on understanding the regulation and function of the JNK and p38 MAPK pathways. Two different strategies were utilized. First, I used molecular and biochemical techniques to examine how MAP2K and MAP3K mediate signaling specificity and to define their role in the MAPK pathway. Second, I used gene targeted disruption studies to determine the in vivo role ofMAP2K and MAP3K in MAPK activation. I specifically used these approaches to examine: (1) docking interactions between p38 MAPK and MAP2K [MKK3 and MKK6 (Chapter II)]; (2) the differential activation of p38 MAPK by MAP2K [MKK3, MKK4, and MKK6 (Chapter III)]; and (3) the selective involvement of the mixed lineage kinase (MLK) group of MAP3K in JNK and p38 MAPK activation (Chapter IV and Appendix). In addition, I analyzed the role of the MKK3 and MKK6 MAP2K in cell proliferation and the role of the MLK MAP3K in adipocyte differentiation (Chapter III and Chapter IV). Together, these data provide insight into the regulation and function of the stress-activated MAPK signal transduction pathways
The role of p38 MAPK and its substrates in neuronal plasticity and neurodegenerative disease
A significant amount of evidence suggests that the p38-mitogen-activated protein kinase (MAPK) signalling cascade plays a crucial role in synaptic plasticity and in neurodegenerative diseases. In this review we will discuss the cellular localisation and activation of p38 MAPK and the recent advances on the molecular and cellular mechanisms of its substrates: MAPKAPK 2 (MK2) and tau protein. In particular we will focus our attention on the understanding of the p38 MAPK-MK2 and p38 MAPK-tau activation axis in controlling neuroinflammation, actin remodelling and tau hyperphosphorylation, processes that are thought to be involved in normal ageing as well as in neurodegenerative diseases. We will also give some insight into how elucidating the precise role of p38 MAPK-MK2 and p38 MAPK-tau signalling cascades may help to identify novel therapeutic targets to slow down the symptoms observed in neurodegenerative diseases such as Alzheimer's and Parkinson's disease
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