Mitogen-activated protein kinase p38 and MK2, MK3 and MK5: Ménage à trois or ménage à quatre?

The mitogen-activated protein kinase (MAPK) signalling pathways play pivotal roles in cellular processes such as proliferation, apoptosis, gene regulation, differentiation, and cell motility. The typical mammalian MAPK pathways ERK1/2, JNK, p38^MAPK, and ERK5 operate through a concatenation of three successive phosphorylation events mediated by a MAPK kinase kinase, a MAPK kinase, and a MAPK. MAPKs phosphorylate substrates with distinct functions, including other protein kinases referred to as MAPK-activated protein kinases. One family of related MAPK-activated protein kinases includes MK2, MK3, and MK5. While it is generally accepted that MK2 and MK3 are bona fide substrates for p38^MAPK, the genuineness of MK5 as a p38^MAPK substrate is disputed. This review summarizes the findings pro and contra an authentic p38^MAPK-MK5 relationship, discusses possible explanations for these discrepancies, and proposes experiments that may help to unequivocally clarify whether MK5 is indeed a substrate for p38^MAPK.     

Sec6 enhances cell migration and suppresses apoptosis by elevating the phosphorylation of p38 MAPK,MK2, and HSP27

The signaling axis of p38 mitogen-activated protein kinase (p38 MAPK) and MAPK-activated protein kinase 2 (MK2) is the dominant pathway that leads to heat shock protein 27 (HSP27) phosphorylation. After activation of MK2 by p38 MAPK, HSP27 is phosphorylated and depolymerized by MK2, thereby increasing the cell migration and directly interfering with the apoptotic signaling cascades. Sec6 is one of the components of the exocyst complex that is an evolutionarily conserved 8-protein complex. Even though several studies have demonstrated that Sec6 is involved in various cellular physiological functions, the relationship between Sec6 and HSP27 or p38 MAPK during cell migration and apoptosis remains unclear. In the present study, we observed that Sec6 increased the phosphorylation of p38 MAPK through the activation of MAPK kinase 3/6 (MKK3/6). Moreover, Sec6 knockdown suppressed the phosphorylation of HSP27 at Ser⁷⁸ and Ser⁸² sites via suppression of activated MK2. Furthermore, the reduction of phosphorylated HSP27 or p38 MAPK by Sec6 knockdown suppressed cell migration and promoted apoptosis after treatment with tumor necrosis factor-α and cycloheximide. The present study suggested that Sec6 is involved in the enhancement of cell migration and suppression of apoptosis through the activation of HSP27 or p38 MAPK phosphorylation.     

Modulation of F-actin rearrangement by the cyclic AMP/cAMP-dependent protein kinase (PKA) pathway is mediated by MAPK-activated protein kinase 5 and requires PKA-induced nuclear export of MK5

The MAPK-activated protein kinases belong to the Ca2+/calmodulin-dependent protein kinases. Within this group, MK2, MK3, and MK5 constitute three structurally related enzymes with distinct functions. Few genuine substrates for MK5 have been identified, and the only known biological role is in ras-induced senescence and in tumor suppression. Here we demonstrate that activation of cAMP-dependent protein kinase (PKA) or ectopic expression of the catalytic subunit Calpha in PC12 cells results in transient nuclear export of MK5, which requires the kinase activity of both Calpha and MK5 and the ability of Calpha to enter the nucleus. Calpha and MK5, but not MK2, interact in vivo, and Calpha increases the kinase activity of MK5. Moreover, Calpha augments MK5 phosphorylation, but not MK2, whereas MK5 does not seem to phosphorylate Calpha. Activation of PKA can induce actin filament accumulation at the plasma membrane and formation of actin-based filopodia. We demonstrate that small interfering RNA-triggered depletion of MK5 interferes with PKA-induced F-actin rearrangement. Moreover, cytoplasmic expression of an activated MK5 variant is sufficient to mimic PKA-provoked F-actin remodeling. Our results describe a novel interaction between the PKA pathway and MAPK signaling cascades and suggest that MK5, but not MK2, is implicated in PKA-induced microfilament rearrangement.     

PKA-induced F-actin rearrangement requires phosphorylation of Hsp27 by the MAPKAP kinase MK5

Mitogen-activated protein kinase (MAPK) pathways can play a role in F-actin dynamics. In particular, the p38 MAPK/MAPK-activated protein kinase 2 (MK2)/heat shock protein 27 (Hsp27) pathway is involved in F-actin alternations. Previously, we showed that MK5 is implicated in F-actin rearrangement induced by the cAMP/cAMP-dependent protein kinase pathway in PC12 cells, while others found Hsp27 to be a good in vitro MK5 substrate. Here we demonstrate that MK5 can specifically interact with Hsp27 in vivo and can induce phosphorylation at serine residues 78 and 82 in cells. siRNA-mediated depletion of Hsp27 protein levels, as well as overexpression of the non-phosphorylatable Hsp27-3A mutant prevented forskolin-induced F-actin reorganization. While ectopic expression of a constitutive active MK5 mutant was sufficient to induce F-actin rearrangement in PC12 cells, co-expression of Hsp27-3A could ablate this process. Our results imply that MK5 is involved in Hsp27-controlled F-actin dynamics in response to activation of the cAMP-dependent protein kinase pathway. These findings render the MK5/Hsp27 connection into a putative therapeutic target for conditions with aberrant Hsp27 phosphorylation such as metastasis, cardiovascular diseases, muscle atrophy, autoimmune skin disease and neuropathology.     

p38 mitogen-activated protein kinase and mitogen-activated protein kinase-activated protein kinase 2 (MK2) signaling in atrophic and hypertrophic denervated mouse skeletal muscle

Background

p38 mitogen-activated protein kinase has been implicated in both skeletal muscle atrophy and hypertrophy. T317 phosphorylation of the p38 substrate mitogen-activated protein kinase-activated protein kinase 2 (MK2) correlates with muscle weight in atrophic and hypertrophic denervated muscle and may influence the nuclear and cytoplasmic distribution of p38 and/or MK2. The present study investigates expression and phosphorylation of p38, MK2 and related proteins in cytosolic and nuclear fractions from atrophic and hypertrophic 6-days denervated skeletal muscles compared to innervated controls.

Methods

Expression and phosphorylation of p38, MK2, Hsp25 (heat shock protein25_rodent/27_human, Hsp25/27) and Hsp70 protein expression were studied semi-quantitatively using Western blots with separated nuclear and cytosolic fractions from innervated and denervated hypertrophic hemidiaphragm and atrophic anterior tibial muscles. Unfractionated innervated and denervated atrophic pooled gastrocnemius and soleus muscles were also studied.

Results

No support was obtained for a differential nuclear/cytosolic localization of p38 or MK2 in denervated hypertrophic and atrophic muscle. The differential effect of denervation on T317 phosphorylation of MK2 in denervated hypertrophic and atrophic muscle was not reflected in p38 phosphorylation nor in the phosphorylation of the MK2 substrate Hsp25. Hsp25 phosphorylation increased 3-30-fold in all denervated muscles studied. The expression of Hsp70 increased 3-5-fold only in denervated hypertrophic muscles.

Conclusions

The study confirms a differential response of MK2 T317 phosphorylation in denervated hypertrophic and atrophic muscles and suggests that Hsp70 may be important for this. Increased Hsp25 phosphorylation in all denervated muscles studied indicates a role for factors other than MK2 in the regulation of this phosphorylation.

     

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.     

Absence of a human DnaJ protein hTid-1S correlates with aberrant actin cytoskeleton organization in lesional psoriatic skin

The biochemical mechanism by which the human tumorous imaginal disc1(S) (hTid-1(S)) interferes with actin cytoskeleton organization in keratinocytes of human skin epidermis was investigated. We found that hTid-1, specifically hTid-1(S), interacts with MK5, a p38-regulated/activated protein kinase, and inhibits the protein kinase activity of MK5 that phosphorylates heat shock protein HSP27 in cultured HeLa cells. Thus, hTid-1(S) expression inhibits the phosphorylation of HSP27 known to play important roles in F-actin polymerization and actin cytoskeleton organization. The interplay between MK5/HSP27 signaling and hTid-1(S) expression was supported by the inhibition of HSP27 phosphorylation and MK5 activity in HeLa cells in response to hypoxia during which hTid-1(S) expression was down-regulated. We also found that overexpression of hTid-1(S) results in the inhibition of HSP27 phosphorylation, F-actin polymerization, and actin cytoskeleton organization in transduced HaCaT keratinocytes. This study further proposes that the loss of hTid-1(S) expression in the basal layer of skin epidermis correlates with the enhanced HSP27 phosphorylation, keratinocyte hyperproliferation, and excess actin cytoskeleton organization in lesional psoriatic skin.     

14-3-3epsilon inhibits MK5-mediated cell migration by disrupting F-actin polymerization

The signal pathway by which 14-3-3epsilon inhibits cell migration induced by MAPK-activated protein kinase 5 (MK5) was investigated in cultured HeLa cells. Both in vivo and in vitro analyses have revealed that 14-3-3epsilon interacts with MK5. 14-3-3epsilon bound to MK5 inhibits the phosphorylation of HSP27, a known substrate of MK5. Disturbance of actin cytoskeleton organization by 14-3-3epsilon was shown in transfected cells transiently expressing 14-3-3epsilon as well as established cells stably expressing 14-3-3epsilon. Moreover, overexpression of 14-3-3epsilon resulted in the inhibition of cell migration induced by MK5 overexpression or TNFalpha treatment. Our results suggest that 14-3-3epsilon bound to MK5 inhibits cell migration by inhibiting the phosphorylation of HSP27 whose phosphorylation regulates F-actin polymerization, actin cytoskeleton organization and subsequent actinfilament dynamics.     

The p38/MK2/Hsp25 pathway is required for BMP-2-induced cell migration

Background

Bone morphogenetic proteins (BMPs) have been shown to participate in the patterning and specification of several tissues and organs during development and to regulate cell growth, differentiation and migration in different cell types. BMP-mediated cell migration requires activation of the small GTPase Cdc42 and LIMK1 activities. In our earlier report we showed that activation of LIMK1 also requires the activation of PAKs through Cdc42 and PI3K. However, the requirement of additional signaling is not clearly known.

Methodology/Principal Findings

Activation of p38 MAPK has been shown to be relevant for a number of BMP-2′s physiological effects. We report here that BMP-2 regulation of cell migration and actin cytoskeleton remodelling are dependent on p38 activity. BMP-2 treatment of mesenchymal cells results in activation of the p38/MK2/Hsp25 signaling pathway downstream from the BMP receptors. Moreover, chemical inhibition of p38 signaling or genetic ablation of either p38α or MK2 blocks the ability to activate the downstream effectors of the pathway and abolishes BMP-2-induction of cell migration. These signaling effects on p38/MK2/Hsp25 do not require the activity of either Cdc42 or PAK, whereas p38/MK2 activities do not significantly modify the BMP-2-dependent activation of LIMK1, measured by either kinase activity or with an antibody raised against phospho-threonine 508 at its activation loop. Finally, phosphorylated Hsp25 colocalizes with the BMP receptor complexes in lamellipodia and overexpression of a phosphorylation mutant form of Hsp25 is able to abolish the migration of cells in response to BMP-2.

Conclusions

These results indicate that Cdc42/PAK/LIMK1 and p38/MK2/Hsp25 pathways, acting in parallel and modulating specific actin regulatory proteins, play a critical role in integrating responses during BMP-induced actin reorganization and cell migration.     

MAPKAPK-2 modulates p38-MAPK localization and small heat shock protein phosphorylation but does not mediate the injury associated with p38-MAPK activation during myocardial ischemia

MAPKAPK-2 (MK2) is a protein kinase activated downstream of p38-MAPK which phosphorylates the small heat shock proteins HSP27 and αB crystallin and modulates p38-MAPK cellular distribution. p38-MAPK activation is thought to contribute to myocardial ischemic injury; therefore, we investigated MK2 effects on ischemic injury and p38 cellular localization using MK2-deficient mice (KO). Immunoblotting of extracts from Langendorff-perfused hearts subjected to aerobic perfusion or global ischemia or reperfusion showed that the total and phosphorylated p38 levels were significantly lower in MK2^−/− compared to MK2^+/+ hearts at baseline, but the ratio of phosphorylated/total p38 was similar. These results were confirmed by cellular fractionation and immunoblotting for both cytosolic and nuclear compartments. Furthermore, HSP27 and αB crsytallin phosphorylation were reduced to baseline in MK2^−/− hearts. On semiquantitative immunofluorescence laser confocal microscopy of hearts during aerobic perfusion, the mean total p38 fluorescence was significantly higher in the nuclear compared to extranuclear (cytoplasmic, sarcomeric, and sarcolemmal compartments) in MK2^+/+ hearts. However, although the increase in phosphorylated p38 fluorescence intensity in all compartments following ischemia in MK2^+/+ hearts was lost in MK2^−/− hearts, it was basally elevated in nuclei of MK2^−/− hearts and was similar to that seen during ischemia in MK2^+/+ hearts. Despite these differences, similar infarct volumes were recorded in wild-type MK2^+/+ and MK2^−/− hearts, which were decreased by the p38 inhibitor SB203580 (1 μM) in both genotypes. In conclusion, p38 MAPK-induced myocardial ischemic injury is not modulated by MK2. However, the absence of MK2 perturbs the cellular distribution of p38. The preserved nuclear distribution of active p38 MAPK in MK2^−/− hearts and the conserved response to SB203580 suggests that activation of p38 MAPK may contribute to injury independently of MK2. Diana A Gorog and Rita I Jabr made equal contributions to this work.     

Vascular endothelial growth factor receptor-2 couples cyclo-oxygenase-2 with pro-angiogenic actions of leptin on human endothelial cells

Background

The adipocyte-derived hormone leptin influences the behaviour of a wide range of cell types and is now recognised as a pro-angiogenic and pro-inflammatory factor. In the vasculature, these effects are mediated in part through its direct leptin receptor (ObRb)-driven actions on endothelial cells (ECs) but the mechanisms responsible for these activities have not been established. In this study we sought to more fully define the molecular links between inflammatory and angiogenic responses of leptin-stimulated human ECs.

Methodology/Principal Findings

Immunoblotting studies showed that leptin increased cyclo-oxygenase-2 (COX-2) expression (but not COX-1) in cultured human umbilical vein ECs (HUVEC) through pathways that depend upon activation of both p38 mitogen-activated protein kinase (p38^MAPK) and Akt, and stimulated rapid phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) on Tyr¹¹⁷⁵. Phosphorylation of VEGFR2, p38^MAPK and Akt, and COX-2 induction in cells challenged with leptin were blocked by a specific leptin peptide receptor antagonist. Pharmacological inhibitors of COX-2, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and p38^MAPK abrogated leptin-induced EC proliferation (assessed by quantifying 5-bromo-2′-deoxyuridine incorporation, calcein fluorescence and propidium iodide staining), slowed the increased migration rate of leptin-stimulated cells (in vitro wound healing assay) and inhibited leptin-induced capillary-like tube formation by HUVEC on Matrigel. Inhibition of VEGFR2 tyrosine kinase activity reduced leptin-stimulated p38^MAPK and Akt activation, COX-2 induction, and pro-angiogenic EC responses, and blockade of VEGFR2 or COX-2 activities abolished leptin-driven neo-angiogenesis in a chick chorioallantoic membrane vascularisation assay in vivo.

Conclusions/Significance

We conclude that a functional endothelial p38^MAPK/Akt/COX-2 signalling axis is required for leptin''s pro-angiogenic actions and that this is regulated upstream by ObRb-dependent activation of VEGFR2. These studies identify a new function for VEGFR2 as a mediator of leptin-stimulated COX-2 expression and angiogenesis and have implications for understanding leptin''s regulation of the vasculature in both non-obese and obese individuals.     

Mitogen Activated Protein Kinase Activated Protein Kinase 2 Regulates Actin Polymerization and Vascular Leak in Ventilator Associated Lung Injury

Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury. Wild type and MK2^−/− mice received mechanical ventilation with high (20 ml/kg) or low (7 ml/kg) tidal volumes up to 4 hrs, after which lungs were harvested for immunohistochemistry, immunoblotting and lung permeability assays. High tidal volume mechanical ventilation resulted in significant phosphorylation of p38 MAP kinase, MK2, HSP25, actin polymerization, and an increase in pulmonary vascular permeability in wild type mice as compared to spontaneous breathing or low tidal volume mechanical ventilation. However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability. Finally, MK2^−/− mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HV_T MV. Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling.     

Cytoskeletal changes in hypoxic pulmonary endothelial cells are dependent on MAPK-activated protein kinase MK2

Exposure to hypoxia causes structural changes in the endothelial cell layer that alter its permeability and its interaction with leukocytes and platelets. One of the well characterized cytoskeletal changes in response to stress involves the reorganization of the actin cytoskeleton and the formation of stress fibers. This report describes cytoskeletal changes in pulmonary microvascular endothelial cells in response to hypoxia and potential mechanisms involved in this process. The hypoxia-induced actin redistribution appears to be mediated by components downstream of MAPK p38, which is activated in pulmonary endothelial cells in response to hypoxia. Our results indicate that kinase MK2, which is a substrate of p38, becomes activated by hypoxia, leading to the phosphorylation of one of its substrates, HSP27. Because HSP27 phosphorylation is known to alter actin distribution in response to other stimuli, we postulate that it also causes the actin redistribution observed in hypoxia. This notion is supported by the observations that similar actin redistribution occurs in cells overexpressing constitutively active MK2 or phosphomimicking HSP27 mutant. Overexpressing dominant negative MK2 blocks the effects of hypoxia on the actin cytoskeleton. Taken together these results indicate that hypoxia stimulates the p38-MK2-HSP27 pathway leading to significant alteration in the actin cytoskeleton.     

Characterization of a novel MK3 splice variant from murine ventricular myocardium

p38 MAP kinase (MAPK) isoforms α, β, and γ, are expressed in the heart. p38α appears pro-apoptotic whereas p38β is pro-hypertrophic. The mechanisms mediating these divergent effects are unknown; hence elucidating the downstream signaling of p38 should further our understanding. Downstream effectors include MAPK-activated protein kinase (MK)-3, which is expressed in many tissues including skeletal muscles and heart. We cloned full-length MK3 (MK3.1, 384 aa) and a novel splice variant (MK3.2, 266 aa) from murine heart. For MK3.2, skipping of exons 8 and 9 resulted in a frame-shift in translation of the first 85 base pairs of exon 10 followed by an in-frame stop codon. Of 3 putative phosphorylation sites for p38 MAPK, only Thr-203 remained functional in MK3.2. In addition, MK3.2 lacked nuclear localization and export signals. Quantitative real-time PCR confirmed the presence of these mRNA species in heart and skeletal muscle; however, the relative abundance of MK3.2 differed. Furthermore, whereas total MK3 mRNA was increased, the relative abundance of MK3.2 mRNA decreased in MK2^?/? mice. Immunoblotting revealed 2 bands of MK3 immunoreactivity in ventricular lysates. Ectopically expressed MK3.1 localized to the nucleus whereas MK3.2 was distributed throughout the cell; however, whereas MK3.1 translocated to the cytoplasm in response to osmotic stress, MK3.2 was degraded. The p38α/β inhibitor SB203580 prevented the degradation of MK3.2. Furthermore, replacing Thr-203 with alanine prevented the loss of MK3.2 following osmotic stress, as did pretreatment with the proteosome inhibitor MG132. In vitro, GST-MK3.1 was strongly phosphorylated by p38α and p38β, but a poor substrate for p38δ and p38γ. GST-MK3.2 was poorly phosphorylated by p38α and p38β and not phosphorylated by p38δ and p38γ. Hence, differential regulation of MKs may, in part, explain diverse downstream effects mediated by p38 signaling.     

A redox microenvironment is essential for MAPK-dependent secretion of pro-inflammatory cytokines: modulation by glutathione (GSH/GSSG) biosynthesis and equilibrium in the alveolar epithelium

The characterization of oxidant (glutathione)-dependent regulation of MAPK^p38/RK-mediated TNF-α secretion was undertaken in vitro, and the ramifications of the influence of a redox microenvironment were unraveled. Intermittent exposure of alveolar epithelial cells (FATEII) to LPS (endotoxin) transiently and temporally induced the expression of MAPK^p38/RK. This upregulation was associated with the activation of MAPKAP-K₂, manifested by the specific phosphorylation of the downstream heat-shock protein (Hsp)-27. Selective blockading of the MAPK^p38/RK pathway using the pyridinyl imidazole SB-203580 abrogated the LPS-dependent release of TNF-α. N-acetyl-l-cysteine (NAC), a precursor of glutathione, reduced TNF-α secretion and increased [GSH]. Conversely, l-buthionine-(S,R)-sulfoximine (BSO), an irreversible inhibitor of γ-glutamylcysteine synthetase (γ-GCS), the rate-limiting enzyme in the pathway mediating GSH biosynthesis, augmented the secretion of TNF-α and [GSSG] accumulation. Whereas NAC abrogated the phosphorylation of MAPK^p38/RK, BSO reversibly amplified this effect. Furthermore, intermittent exposure of FATEII cells to the exogenous oxidants X/XO and H₂O₂ upregulated the secretion of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α; this upregulation was correlated with increasing activity of key glutathione-related enzymes, closely involved with maintaining the cyclic GSH/GSSG equilibrium. These results indicate that a redox microenvironment plays a major role in regulating MAPK-dependent production of cytokines in the alveolar epithelium.     

Mechanism of cell death of rat cardiac fibroblasts induced by serum depletion

Serum starvation has recently been shown to cause cell death of cardiac fibroblasts and increased synthesis of extracellular matrix proteins in the surviving cells. In the present study, events occurring in the dying cells were investigated. Cultured adult rat cardiac fibroblasts were exposed to serum-free medium. Cell number was measured using a Coulter Counter Channelyzer. The activity of the extracellular signal-regulated or mitogen-activated protein kinases (ERK1/2, p42/p44^MAPK), the p38 kinase (p38^MAPK), the c-Jun N-terminal kinases (p46/p54^JNK), and Akt kinase was assessed by Western blotting and phospho-specific antibodies. Caspase 7-cleavage was investigated by Western blotting and specific antibodies. Caspase 3 activity was measured by detection of its cleaved substrate. The appearance of necrosis was studied by inclusion of trypan blue. Apoptosis was assessed by DNA ladder formation. The mRNA expression of Bax and Bcl-2 was investigated by quantitative real-time PCR. Serum withdrawal led to the death of 26% of cultured isolated cardiac fibroblasts during the first 5 h. The activity of the p42/p44^MAPK as well as of Akt kinase was partially reduced. For p46/p54^JNK and p38^MAPK, elevated phosphorylation was measured. Inhibition of p46/p54^JNK and p38^MAPK activity by SB202190 did not affect the decrease in cell number. Cleavage of caspase7 was detected after 90 min. However, no activation of caspase 3 was measured. DNA fragmentation was not found after serum depletion. Trypan blue staining, however, was observed in 16% of the cells after 5 h. The mRNA levels of both Bax and Bcl-2 were increased after 30 min. These results indicate the appearance of necrosis during serum starvation in cardiac fibroblasts. However, some processes typical of apoptosis were also detected.     

Promyelocytic leukemia protein targets MK2 to promote cytotoxicity

Promyelocytic leukemia protein (PML) is a tumor suppressor possessing multiple modes of action, including induction of apoptosis. We unexpectedly find that PML promotes necroptosis in addition to apoptosis, with Pml ^−/− macrophages being more resistant to TNF‐mediated necroptosis than wild‐type counterparts and PML‐deficient mice displaying resistance to TNF‐induced systemic inflammatory response syndrome. Reduced necroptosis in PML‐deficient cells is associated with attenuated receptor‐interacting protein kinase 1 (RIPK1) activation, as revealed by reduced RIPK1[S166] phosphorylation, and attenuated RIPK1‐RIPK3‐MLKL necrosome complex formation. We show that PML deficiency leads to enhanced TNF‐induced MAPK‐activated kinase 2 (MK2) activation and elevated RIPK1[S321] phosphorylation, which suppresses necrosome formation. MK2 inhibitor treatment or MK2 knockout abrogates resistance to cell death induction in PML‐null cells and mice. PML binds MK2 and p38 MAPK, thereby inhibiting p38‐MK2 interaction and MK2 activation. Moreover, PML participates in autocrine production of TNF induced by cellular inhibitors of apoptosis 1 (cIAP1)/cIAP2 degradation, since PML‐knockout attenuates autocrine TNF. Thus, by targeting MK2 activation and autocrine TNF, PML promotes necroptosis and apoptosis, representing a novel tumor‐suppressive activity for PML.