The p38 Pathway Regulates Oxidative Stress Tolerance by Phosphorylation of Mitochondrial Protein IscU

The p38 pathway is an evolutionarily conserved signaling pathway that responds to a variety of stresses. However, the underlying mechanisms are largely unknown. In the present study, we demonstrate that p38b is a major p38 MAPK involved in the regulation of oxidative stress tolerance in addition to p38a and p38c in Drosophila. We further show the importance of MK2 as a p38-activated downstream kinase in resistance to oxidative stresses. Furthermore, we identified the iron-sulfur cluster scaffold protein IscU as a new substrate of MK2 both in Drosophila cells and in mammalian cells. These results imply a new mechanistic connection between the p38 pathway and mitochondria iron-sulfur clusters.     

Endosomal targeting of the phosphoinositide 3-phosphatase MTMR2 is regulated by an N-terminal phosphorylation site

MTMR2 is a member of the myotubularin family of inositol lipid phosphatases, a large protein-tyrosine phosphatase subgroup that is conserved from yeast to humans. Furthermore, the peripheral neuromuscular disease Charcot-Marie Tooth disease type 4B has been attributed to mutations in the mtmr2 gene. Because the molecular mechanisms regulating MTMR2 have been poorly defined, we investigated whether reversible phosphorylation might regulate MTMR2 function. We used mass spectrometry-based methods to identify a high stoichiometry phosphorylation site on serine 58 of MTMR2. Phosphorylation at Ser(58), or a phosphomimetic S58E mutation, markedly decreased MTMR2 localization to endocytic vesicular structures. In contrast, a phosphorylation-deficient MTMR2 mutant (S58A) displayed constitutive localization to early endocytic structures. This localization pattern was accompanied by displacement of a PI(3)P-specific sensor protein and an increase in signal transduction pathways. Thus, MTMR2 phosphorylation is likely to be a critical mechanism by which MTMR2 access to its lipid substrate(s) is temporally and spatially regulated, thereby contributing to the control of downstream endosome maturation events.     

Phosphorylated alpha-synuclein at Ser-129 is targeted to the proteasome pathway in a ubiquitin-independent manner

α-Synuclein (a-Syn) is a major component of fibrillar aggregates in Lewy bodies (LBs), a characteristic hallmark of Parkinson disease. Almost 90% of a-Syn deposited in LBs is phosphorylated at Ser-129. However, the role of Ser-129-phosphorylated a-Syn in the biogenesis of LBs remains unclear. Here, we investigated the metabolism of Ser-129-phosphorylated a-Syn. In SH-SY5Y cells, inhibition of protein phosphatase 2A/1 by okadaic acid, and inhibition of the proteasome pathway by MG132 or lactacystin accumulated Ser-129-phosphorylated a-Syn. However, these inhibitions did not alter the amounts of total a-Syn within the observation time. Inhibition of the autophagy-lysosome pathway by 3-methyladenine or chloroquine accumulated Ser-129-phosphorylated a-Syn in parallel to total a-Syn during longer incubations. Experiments using cycloheximide showed that Ser-129-phosphorylated a-Syn diminished rapidly (t(½) = 54.9 ± 6.4 min), in contrast to the stably expressed total a-Syn. The short half-life of Ser-129-phosphorylated a-Syn was blocked by MG132 to a greater extent than okadaic acid. In rat primary cortical neurons, either MG132, lactacystin, or okadaic acid accumulated Ser-129-phosphorylated a-Syn. Additionally, we did not find that phosphorylated a-Syn was ubiquitinated in the presence of proteasome inhibitors. These data show that Ser-129-phosphorylated a-Syn is targeted to the proteasome pathway in a ubiquitin-independent manner, in addition to undergoing dephosphorylation. The proteasome pathway may play a role in the biogenesis of Ser-129-phosphorylated a-Syn-rich LBs.     

Cell type-specific activation of p38 MAPK in the brain regions of hypoxic preconditioned mice

Activation of p38 mitogen-activated protein kinase (p38 MAPK) has been implicated as a mechanism of ischemia/hypoxia-induced cerebral injury. The current study was designed to explore the involvement of p38 MAPK in the development of cerebral hypoxic preconditioning (HPC) by observing the changes in dual phosphorylation (p-p38 MAPK) at threonine180 and tyrosine182 sites, protein expression, and cellular distribution of p-p38 MAPK in the brain of HPC mice. We found that the p-p38 MAPK levels, not protein expression, increased significantly (p < 0.05) in the regions of frontal cortex, hippocampus, and hypothalamus of mice in response to repetitive hypoxic exposure (H1–H6, n = 6 for each group) when compared to values of the control normoxic group (H0, n = 6) using Western blot analysis. Similar results were also confirmed by an immunostaining study of the p-p38 MAPK location in the frontal cortex, hippocampus, and hypothalamus of mice from HPC groups. To further define the cell type of p-p38 MAPK positive cells, we used a double-labeled immunofluorescent staining method to co-localize p-p38 MAPK with neurofilaments heavy chain (NF-H, neuron-specific marker), S100 (astrocyte-specific marker), and CD11b (microglia-specific maker), respectively. We found that the increased p-p38 MAPK occurred in microglia of cortex and hippocampus, as well as in neurons of hypothalamus of HPC mice. These results suggest that the cell type-specific activation of p38 MAPK in the specific brain regions might contribute to the development of cerebral HPC mechanism in mice.     

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.     

Cocaine up-regulation of the norepinephrine transporter requires threonine 30 phosphorylation by p38 mitogen-activated protein kinase

The norepinephrine (NE) transporter (NET) regulates NE signaling by rapidly clearing synaptic NE. Cocaine binds NET and modulates NE transport. These actions contribute to rewarding effects and abuse liability of cocaine. Activation of mitogen-activated protein kinase (MAPK) cascades is implicated in cocaine-induced neuroadaptations. However, the role of MAPK and the mechanisms involved in cocaine modulation of NET are not clear. Acute intra-peritoneal injections of cocaine (20 mg/kg body weight) to rats resulted in increased NE uptake by prefrontal cortex (PFC) synaptosomes with a parallel increase in the surface expression of endogenous NET. Cocaine also enhanced the immunoreactivity of phospho-p38 MAPK in the PFC synaptosomes without affecting the total p38 MAPK. In vitro cocaine (30-50 μM) treatment of rat PFC synaptosomes increased native NET function, surface expression, and phosphorylation in a manner sensitive to p38 MAPK inhibition by PD169316. We next examined cocaine-elicited effects on wild-type human NET (hNET) expressed heterologously in human placental trophoblast cells to gain more insights into the mechanisms involved. Cocaine treatment of hNET expressing human placental trophoblast cells up-regulated the function, surface expression, and phosphorylation of hNET in a PD169316-sensitive manner. In addition, cocaine inhibited constitutive endocytosis of hNET. Mutational analysis of serine and threonine residues revealed that substitution of threonine 30, located at the amino terminus of hNET with alanine (T30A-hNET), abolished cocaine-induced up-regulation of NET function, surface expression, and phosphorylation. Furthermore, cocaine did not alter T30A-hNET endocytosis. These studies identify a novel molecular mechanism that cocaine-activated p38 MAPK-mediated phosphorylation of NET-T30 dictates surface NET availability, and hence, NE transport.     

mTOR complex 2 targets Akt for proteasomal degradation via phosphorylation at the hydrophobic motif

The protein kinase Akt (also known as protein kinase B) is a critical signaling hub downstream of various cellular stimuli such as growth factors that control cell survival, growth, and proliferation. The activity of Akt is tightly regulated, and the aberrant activation of Akt is associated with diverse human diseases including cancer. Although it is well documented that the mammalian target of rapamycin complex 2 (mTORC2)-dependent phosphorylation of the Akt hydrophobic motif (Ser-473 in Akt1) is essential for full Akt activation, it remains unclear whether this phosphorylation has additional roles in regulating Akt activity. In this study, we found that abolishing Akt Ser-473 phosphorylation stabilizes Akt following agonist stimulation. The Akt Ser-473 phosphorylation promotes a Lys-48-linked polyubiquitination of Akt, resulting in its rapid proteasomal degradation. Moreover, blockade of this proteasomal degradation pathway prolongs agonist-induced Akt activation. These data reveal that mTORC2 plays a central role in regulating the Akt protein life cycle by first stabilizing Akt protein folding through the turn motif phosphorylation and then by promoting Akt protein degradation through the hydrophobic motif phosphorylation. Taken together, this study reveals that the Akt Ser-473 phosphorylation-dependent ubiquitination and degradation is an important negative feedback regulation that specifically terminates Akt activation.     

p38α- and DYRK1A-dependent phosphorylation of caspase-9 at an inhibitory site in response to hyperosmotic stress

The cysteine aspartyl protease caspase-9 is a critical component of the intrinsic apoptotic pathway. Activation of caspase-9 is inhibited by phosphorylation at Thr125, which is catalysed by the mitogen-activated protein kinases (MAPKs) ERK1/2 in response to growth factors, by the cyclin-dependent protein kinase CDK1-cyclin B1 during mitosis, and at a basal level by the dual-specificity tyrosine-phosphorylation regulated protein kinase DYRK1A. Here we show that inhibitory phosphorylation of caspase-9 at Thr125 is induced in mammalian cells by hyperosmotic stress. This response does not require ERK1/2 or ERK5, but it is diminished by ablation of DYRK1A expression by siRNA or chemical inhibition of DYRK1A by harmine. Phosphorylation of Thr125 in response to hyperosmotic stress is also reduced by chemical inhibition of p38 MAPK and is abolished in p38α^−/− mouse embryonic fibroblasts. These results show that both DYRK1A and p38α play roles in the inhibitory phosphorylation of caspase-9 following hyperosmotic stress and suggest a functional interaction between these protein kinases. Phosphorylation of caspase-9 at Thr125 may restrain apoptosis during the acute response to hyperosmotic stress.     

大鼠脑内远位触液神经元p38丝裂原活化蛋白激酶的分布及在噪声应激时的表达

目的：观察脑内远位触液神经元内p-p38丝裂原活化蛋白激酶（MAPK）的分布及其在噪声应激时的表达。方法：用霍乱毒素亚单位B与辣根过氧化物酶复合物（CB-HRP）标记和免疫组织化学相结合的双重标记技术．观察SD大鼠脑实质内远位触液神经元中p-p38MAPK的分布：进一步制作噪声应激动物模型,观察噪声应激后该类神经元中p-p38MAPK的表达变化。结果：在脑干的特定部位恒定出现被CB-HRP标记的两组神经细胞簇,其他脑区未见CB-HRP标记神经细胞簇。不予应激刺激,该细胞簇内仅有个别神经元见有CB-HRP／p—p38MAPK;噪声应激刺激1d时,上述特定部位细胞簇的CB-HRP／p-p38MAPK双重标记神经元数目没有明显变化;噪音应激刺激5d时,CB-HRP／p—p38MAPK双重标记神经元数目较对照组显著增多（P〈0．05）;噪音应激刺激10d时CB-HRP／p—p38MAPK双重标记神经元数目较对照组显著增多（P〈0．05）;噪音应激刺激20d时,CB-HRP／p—p38MAPK双重标记神经元数目较对照组显著增多（P〈0．01）：结论：在脑干特定部位恒定存在的两组被CBHRP标记的细胞团为远位触液神经元,其中少数触液神经元有p-p38MAPK表达,且当给予动物噪声应激刺激时,p-p38MAPK免疫阳性神经元和CB-HRP／p—p38MAPK双重标记神经元数量显著增加,提示脑实质内的这种远位触液神经元中的P—p38MAPK可能参与了机体对噪声应激的信息传递或调控,其作用随应激天数增加而日趋增强．     

p38 MAP Kinase and MAPKAP Kinases MK2/3 Cooperatively Phosphorylate Epithelial Keratins

The MAPK-activated protein kinases (MAPKAP kinases) MK2 and MK3 are directly activated via p38 MAPK phosphorylation, stabilize p38 by complex formation, and contribute to the stress response. The list of substrates of MK2/3 is increasing steadily. We applied a phosphoproteomics approach to compare protein phosphorylation in MK2/3-deficient cells rescued or not by ectopic expression of MK2. In addition to differences in phosphorylation of the known substrates of MK2, HSPB1 and Bag-2, we identified strong differences in phosphorylation of keratin 8 (K8). The phosphorylation of K8-Ser⁷³ is catalyzed directly by p38, which in turn shows MK2-dependent expression. Notably, analysis of small molecule p38 inhibitors on K8-Ser⁷³ phosphorylation also demonstrated reduced phosphorylations of keratins K18-Ser⁵² and K20-Ser¹³ but not of K8-Ser⁴³¹ or K18-Ser³³. Interestingly, K18-Ser⁵² and K20-Ser¹³ are not directly phosphorylated by p38 in vitro, but by MK2. Furthermore, anisomycin-stimulated phosphorylations of K20-Ser¹³ and K18-Ser⁵² are inhibited by small molecule inhibitors of both p38 and MK2. MK2 knockdown in HT29 cells leads to reduced K20-Ser¹³ phosphorylation, which further supports the notion that MK2 is responsible for K20 phosphorylation in vivo. Physiologic relevance of these findings was confirmed by differences of K20-Ser¹³ phosphorylation between the ileum of wild-type and MK2/3-deficient mice and by demonstrating p38- and MK2-dependent mucin secretion of HT29 cells. Therefore, MK2 and p38 MAPK function in concert to phosphorylate K8, K18, and K20 in intestinal epithelia.     

2-Deoxy-D-ribose inhibits hypoxia-induced apoptosis by suppressing the phosphorylation of p38 MAPK

An angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-d-ribose, a degradation product of thymidine generated by TP enzymatic activity, partially prevented hypoxia-induced apoptosis. 2-Deoxy-d-ribose inhibited hypoxia-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) but not c-jun NH(2)-terminal kinase/stress-activated protein kinase in human leukemia HL-60 cells. 2-Deoxy-d-ribose also suppressed the levels of Bax attached to mitochondria under hypoxic conditions. SB203580, a specific inhibitor of the p38 MAPK, suppressed the hypoxia-induced apoptosis of HL-60 cells. These findings suggest that one of the molecular bases for resistance to hypoxia-induced apoptosis conferred by 2-deoxy-d-ribose is the inhibition of the p38 signaling pathway. The expression levels of TP are elevated in many malignant solid tumors and thus the 2-deoxy-d-ribose generated by TP in these tumors may play an important role in tumor progression by preventing hypoxia-induced apoptosis.     

Phosphorylation of Raptor by p38beta participates in arsenite-induced mammalian target of rapamycin complex 1 (mTORC1) activation

Cell growth is influenced by environmental stress. Mammalian target of rapamycin (mTOR), the central regulator of cell growth, can be positively or negatively regulated by various stresses through different mechanisms. The p38 MAP kinase pathway is essential in cellular stress responses. Activation of MK2, a downstream kinase of p38α, enhances mTOR complex 1 (mTORC1) activity by preventing TSC2 from inhibiting mTOR activation. The p38β-PRAK cascade targets Rheb to inhibit mTORC1 activity upon glucose depletion. Here we show the activation of p38β participates in activation of mTOR complex 1 (mTORC1) induced by arsenite but not insulin, nutrients, anisomycin, or H(2)O(2). Arsenite treatment of cells activates p38β and induces interaction between p38β and Raptor, a regulatory component of mTORC1, resulting in phosphorylation of Raptor on Ser(863) and Ser(771). The phosphorylation of Raptor on these sites enhances mTORC1 activity, and contributes largely to arsenite-induced mTORC1 activation. Our results shown here and in previous work demonstrate that the p38 pathway can regulate different components of the mTORC1 pathway, and that p38β can target different substrates to either positively or negatively regulate mTORC1 activation when a cell encounters different environmental stresses.