Naringenin targets ERK2 and suppresses UVB‐induced photoaging

A number of natural phytochemicals have anti‐photoaging properties that appear to be mediated through the inhibition of matrix metalloproteinase‐1 (MMP‐1) expression, but their direct target molecule(s) and mechanism(s) remain unclear. We investigated the effect of naringenin, a major flavonoid found in citrus, on UVB‐induced MMP‐1 expression and identified its direct target. The HaCaT human skin keratinocyte cell line and 3‐dimensional (3‐D) human skin equivalent cultures were treated or not treated with naringenin for 1 hr before exposure to UVB. The mechanism and target(s) of naringenin were analysed by kinase assay and multiplex molecular assays. Dorsal skins of hairless mice were exposed to UVB 3 times per week, with a dose of irradiation that was increased weekly by 1 minimal erythema dose (MED; 45 mJ/cm²) to 4 MED over 15 weeks. Wrinkle formation, water loss and water content were then assessed. Naringenin suppressed UVB‐induced MMP‐1 expression and AP‐1 activity, and strongly suppressed UVB‐induced phosphorylation of Fos‐related antigen (FRA)‐1 at Ser265. Importantly, UVB irradiation‐induced FRA1 protein stability was reduced by treatment with naringenin, as well as with a mitogen‐activated protein kinase (MEK) inhibitor. Naringenin significantly suppressed UVB‐induced extracellular signal‐regulated kinase 2 (ERK2) activity and subsequently attenuated UVB‐induced phosphorylation of p90^RSK by competitively binding with ATP. Constitutively active MEK (CA‐MEK) increased FRA1 phosphorylation and expression and also induced MMP‐1 expression, whereas dominant‐negative ERK2 (DN‐ERK2) had opposite effects. U0126, a MEK inhibitor, also decreased FRA1 phosphorylation and expression as well as MMP‐1 expression. The photoaging data obtained from mice clearly demonstrated that naringenin significantly inhibited UVB‐induced wrinkle formation, trans‐epidermal water loss and MMP‐13 expression. Naringenin exerts potent anti‐photoaging effects by suppressing ERK2 activity and decreasing FRA1 stability, followed by down‐regulation of AP‐1 transactivation and MMP‐1 expression.     

Verbascoside down‐regulates some pro‐inflammatory signal transduction pathways by increasing the activity of tyrosine phosphatase SHP‐1 in the U937 cell line

Polyphenols are the major components of many traditional herbal remedies, which exhibit several beneficial effects including anti‐inflammation and antioxidant properties. Src homology region 2 domain‐containing phosphatase‐1 (SHP‐1) is a redox sensitive protein tyrosine phosphatase that negatively influences downstream signalling molecules, such as mitogen‐activated protein kinases, thereby inhibiting inflammatory signalling induced by lipopolysaccharide (LPS). Because a role of transforming growth factor β‐activated kinase‐1 (TAK1) in the upstream regulation of JNK molecule has been well demonstrated, we conjectured that SHP‐1 could mediate the anti‐inflammatory effect of verbascoside through the regulation of TAK‐1/JNK/AP‐1 signalling in the U937 cell line. Our results demonstrate that verbascoside increased the phosphorylation of SHP‐1, by attenuating the activation of TAK‐1/JNK/AP‐1 signalling. This leads to a reduction in the expression and activity of both COX and NOS. Moreover, SHP‐1 depletion deletes verbascoside inhibitory effects on pro‐inflammatory molecules induced by LPS. Our data confirm that SHP‐1 plays a critical role in restoring the physiological mechanisms of inducible proteins such as COX2 and iNOS, and that the down‐regulation of TAK‐1/JNK/AP‐1 signalling by targeting SHP‐1 should be considered as a new therapeutic strategy for the treatment of inflammatory diseases.     

Chlorogenic acid: A potent molecule that protects cardiomyocytes from TNF‐α–induced injury via inhibiting NF‐κB and JNK signals

The traditional Chinese herb Lonicerae Japonicae Flos has shown significant clinical benefits in the treatment of heart failure, but the mechanism remains unclear. As the main active ingredient found in the plasma after oral administration of Lonicerae Japonicae Flos, chlorogenic acid (CGA) has been reported to possess anti‐inflammatory, anti‐oxidant and anti‐apoptosis function. We firstly confirmed the cardioprotective effects of CGA in transverse aortic constriction (TAC)‐induced heart failure mouse model, through mitigating the TNF‐α–induced toxicity. We further used TNF‐α‐induced cardiac injury in human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) to elucidate the underlying mechanisms. CGA pre‐treatment could reverse TNF‐α–induced cellular injuries, including improved cell viability, increased mitochondrial membrane potential and inhibited cardiomyocytes apoptosis. We then examined the NF‐κB/p65 and major mitogen‐activated protein kinases (MAPKs) signalling pathways involved in TNF‐α–induced apoptosis of hiPSC‐CMs. Importantly, CGA can directly inhibit NF‐κB signal by suppressing the phosphorylation of NF‐κB/p65. As for the MAPKs, CGA suppressed the activity of only c‐Jun N‐terminal kinase (JNK), but enhanced extracellular signal‐regulated kinase1/2 (ERK1/2) and had no effect on p38. In summary, our study revealed that CGA has profound cardioprotective effects through inhibiting the activation of NF‐κB and JNK pathway, providing a novel therapeutic alternative for prevention and treatment of heart failure.     

Negative regulation of mixed lineage kinase 3 by protein kinase B/AKT leads to cell survival

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates c-jun N-terminal kinase (JNK) and can induce cell death in neurons. By contrast, the activation of phosphatidylinositol 3-kinase and AKT/protein kinase B (PKB) acts to suppress neuronal apoptosis. Here, we report a functional interaction between MLK3 and AKT1/PKBalpha. Endogenous MLK3 and AKT1 interact in HepG2 cells, and this interaction is regulated by insulin. The interaction domain maps to the C-terminal half of MLK3 (amino acids 511-847), and this region also contains a putative AKT phosphorylation consensus sequence. Endogenous JNK, MKK7, and MLK3 kinase activities in HepG2 cells are significantly attenuated by insulin treatment, whereas the phosphatidylinositol 3-kinase inhibitors LY294002 and wortmannin reversed the effect. Finally, MLK3-mediated JNK activation is inhibited by AKT1. AKT phosphorylates MLK3 on serine 674 both in vitro and in vivo. Furthermore, the expression of activated AKT1 inhibits MLK3-mediated cell death in a manner dependent on serine 674 phosphorylation. Thus, these data provide the first direct link between MLK3-mediated cell death and its regulation by a cell survival signaling protein, AKT1.     

Naringin prevents ultraviolet‐B radiation‐induced oxidative damage and inflammation through activation of peroxisome proliferator‐activated receptor γ in mouse embryonic fibroblast (NIH‐3T3) cells

The present study, we investigate the preventive role of naringin, a dietary flavonoid, against ultraviolet‐B (UVB) radiation (280‐320 nm) induced oxidative damage and inflammatory responses in mouse embryonic fibroblast cell lines (NIH‐3T3). In this study, 20 mJ/cm ² of UVB radiation induces cell cytotoxicity, reactive oxygen species (ROS) generation, DNA damage, and antioxidants depletion in NIH‐3T3 cells. Treatment with naringin (60 µM) prior UVB exposure prevented the cell cytotoxicity, ROS generation, DNA damage, and antioxidants depletion in NIH‐3T3 cells. Furthermore, naringin prevents UVB‐induced mitogen‐activated protein kinase families and nuclear factor‐κB (NF‐κB)‐mediated activation of inflammatory factors, that is TNF‐α, IL‐6, IL‐10, and COX‐2 in NIH‐3T3 cells. Peroxisome proliferator‐activated receptor γ (PPARγ) is an anti‐inflammatory agent and it suppressed the UVB‐mediated oxidative and inflammatory responses. In this study, naringin activates PPARγ and prevents inflammatory biomarkers in NIH‐3T3 cells. Thus, naringin prevents UVB‐mediated inflammation and oxidative damage in NIH‐3T3 cells probably over controlling NF‐κB expression and activation of PPARγ.     

Inhibition of MLK3-MKK4/7-JNK1/2 pathway by Akt1 in exogenous estrogen-induced neuroprotection against transient global cerebral ischemia by a non-genomic mechanism in male rats

Numerous studies have demonstrated the neuroprotective effects of estrogen in experimental cerebral ischemia. To investigate molecular mechanisms of estrogen neuroprotection in global ischemia, immunoblotting, immunohistochemistry and Nissel-staining analysis were used. Our results showed that chronic pretreatment with beta-estradiol 3-benzoate (E2) enhanced Akt1 activation and reduced the activation of mixed-lineage kinase 3 (MLK3), mitogen-activated protein kinase kinase 4/7 (MKK4/7), and c-Jun N-terminal kinase 1/2 (JNK1/2) in the hippocampal CA1 subfield during reperfusion after 15 min of global ischemia. In addition, E2 reduced downstream JNK nuclear and non-nuclear components, c-Jun and Bcl-2 phosphorylation and Fas ligand protein expression induced by ischemia/reperfusion. Administration of phosphoinositide 3-kinase (PI3K) inhibitor LY 294,002 prevented both activation of Akt1 and inhibition of MLK3, MKK4/7 and JNK1/2. The interaction between ERalpha and the p85 subunit of PI3K was also examined. E2 and antiestrogen ICI 182,780 promoted and prevented this interaction, respectively. Furthermore, ICI 182,780 blocked both the activation of Akt1 and the inhibition of MLK3, MKK4/7 and JNK1/2. Photomicrographs of cresyl violet-stained brain sections showed that E2 reduced CA1 neuron loss after 5 days of reperfusion, which was abolished by ICI 182,780 and LY 294,002. Our data indicate that in response to estrogen, ERalpha interacts with PI3K to activate Akt1, which may inhibit the MLK3-MKK4/7-JNK1/2 pathway to protect hippocampal CA1 neurons against global cerebral ischemia in male rats.     

A tissue restricted role for the Xenopus Jun N-terminal kinase kinase kinase MLK2 in cement gland and pronephric tubule differentiation

The MLK family of mitogen activated protein kinase kinase kinases (MAPKKK) has been shown to activate Jun N-terminal kinase/stress-activated protein kinase 1 (JNK/SAPK1). However, little is known of the in vivo functions of the MLKs. We have identified a Xenopus laevis MLK that shows highest homology with mammalian MLK2 (62%) and, like MLK2, interacts preferentially with the Rho-family GTPase Rac. xMLK2 was expressed zygotically from late gastrula/early neurula. Surprisingly, this expression was restricted to the cement gland, the brain, and the pronephros. In the differentiating cement gland, xMLK2 expression correlated with cell elongation and the onset of a previously unobserved apoptotic phase, while in the pronephros, expression corresponded with the differentiation and opening of the nephric tubules. Overexpression of xMLK2 in COS7 cells led to a SEK1/MKK4 (MAPKK)-dependent hyperactivation of JNK in response to UV irradiation. xMLK2 was shown to be required for normal cement gland development and pronephric tubule formation using antisense inactivation and a dominant negative xMLK2. The data suggest a novel role for the MLKs as tissue-restricted mediators of signal transduction. They also suggest that tissue-specific responses to common extracellular signals may in part result from the programmed expression of MAPKKKs with differing specificities.     

Novel functions for 2‐phenylbenzimidazole‐5‐sulphonic acid: Inhibition of ovarian cancer cell responses and tumour angiogenesis

In this study, we investigated the effects and molecular mechanisms of 2‐phenylbenzimidazole‐5‐sulphonic acid (PBSA), an ultraviolet B protecting agent used in sunscreen lotions and moisturizers, on ovarian cancer cell responses and tumour angiogenesis. PBSA treatment markedly blocked mitogen‐induced invasion through down‐regulation of matrix metalloproteinase (MMP) expression and activity in ovarian cancer SKOV‐3 cells. In addition, PBSA inhibited mitogen‐induced cell proliferation by suppression of cyclin‐dependent kinases (Cdks), but not cyclins, leading to pRb hypophosphorylation and G₁ phase cell cycle arrest. These anti‐cancer activities of PBSA in ovarian cancer cell invasion and proliferation were mediated by the inhibition of mitogen‐activated protein kinase kinase 3/6‐p38 mitogen‐activated protein kinase (MKK3/6‐p38^MAPK) activity and subsequent down‐regulation of MMP‐2, MMP‐9, Cdk4, Cdk2 and integrin β1, as evidenced by treatment with p38^MAPK inhibitor SB203580. Furthermore, PBSA suppressed the expression and secretion of vascular endothelial growth factor in SKOV‐3 cells, leading to inhibition of capillary‐like tubular structures in vitro and angiogenic sprouting ex vivo. Taken together, our results demonstrate the pharmacological effects and molecular targets of PBSA on modulating ovarian cancer cell responses and tumour angiogenesis, and suggest further evaluation and development of PBSA as a promising chemotherapeutic agent for the treatment of ovarian cancer.     

4‐phenylpyridine suppresses UVB‐induced skin inflammation by targeting c‐Src in vitro and in vivo

Acute or repetitive exposure to ultraviolet (UV) cause disruptions to the skin barrier and subsequent inflammatory skin disease. 4‐phenylpyridine (4‐PP) is a constituent of Brassica campestris L. ssp. Pekinensis and its effect on skin inflammation and molecular target remain unclear. The purpose of this study is to confirm the anti‐inflammatory efficacy of 4‐PP on UVB‐induced skin inflammation in human keratinocytes HaCaT and mouse skin and validation of its molecular target. 4‐PP also attenuated UVB‐induced phosphorylation of p38/mitogen‐activated protein kinase kinase (MKK) 3/6, c‐Jun N‐terminal kinase 1/2, MKK 4/7, extracellular‐signal‐regulated kinase 1/2, mitogen‐activated protein kinase 1/2. Additionally, 4‐PP inhibited UVB‐induced phosphorylation of epidermal growth factor receptor (EGFR) Y1068, Y1045 and 854 residues but not the proto‐oncogene tyrosine‐protein kinase c‐Src. Drug affinity responsive target stability assay revealed that 4‐PP directly binds to c‐Src and inhibits pronase c‐proteolysis. Knockdown of c‐Src inhibited UVB‐induced COX‐2 expression and phosphorylation of MAPKs and EGFR in HaCaT cells. Dorsal treatment of 4‐PP prevented UVB (0.5 J/cm²)‐induced skin thickness, phosphorylation of EGFR and COX‐2 expression in mouse skin. Our findings suggest that 4‐PP can be used as anti‐inflammatory agent with an effect of skin inflammation by inhibiting the COX‐2 expression via suppressing the c‐Src/EGFR/MAPKs signalling pathway.     

CME‐1, a novel polysaccharide,suppresses iNOS expression in lipopolysaccharide‐stimulated macrophages through ceramide‐initiated protein phosphatase 2A activation

CME‐1, a novel water‐soluble polysaccharide purified from Ophiocordyceps sinensis mycelia, has anti‐oxidative, antithrombotic and antitumour properties. In this study, other major attributes of CME‐1, namely anti‐inflammatory and immunomodulatory properties, were investigated. Treating lipopolysaccharide (LPS)‐stimulated RAW 264.7 cells with CME‐1 concentration‐dependently suppressed nitric oxide formation and inducible nitric oxide synthase (iNOS) expression. In the CME‐1‐treated RAW 264.7 cells, LPS‐induced IκBα degradation and the phosphorylation of p65, Akt and mitogen‐activated protein kinases (MAPKs), including extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase and p38, were reduced. Treatment with a protein phosphatase 2A (PP2A)‐specific inhibitor, significantly reversed the CME‐1‐suppressed iNOS expression; IκBα degradation; and p65, Akt and MAPK phosphorylation. PP2A activity up‐regulation and PP2A demethylation reduction were also observed in the cells. Moreover, CME‐1‐induced PP2A activation and its subsequent suppression of LPS‐activated RAW 264.7 cells were diminished by the inhibition of ceramide signals. LPS‐induced reactive oxygen species (ROS) and hydroxyl radical formation were eliminated by treating RAW 264.7 cells with CME‐1. Furthermore, the role of ceramide signalling pathway and anti‐oxidative property were also demonstrated in CME‐1‐mediated inhibition of LPS‐activated primary peritoneal macrophages. In conclusion, CME‐1 suppressed iNOS expression by up‐regulating ceramide‐induced PP2A activation and reducing ROS production in LPS‐stimulated macrophages. CME‐1 is a potential therapeutic agent for treating inflammatory diseases.     

Involvement of c‐Jun N‐terminal kinase and extracellular signal‐regulated kinase 1/2 in EGF‐induced angiogenesis

Angiogenesis is a process during which endothelial cells divide and migrate to form new capillaries from the preexisting blood vessels. The present study was designed to investigate whether MAPKs (mitogen‐activated protein kinases) play crucial roles in regulating EGF (epidermal growth factor)‐induced endothelial cell angiogenesis. Our results showed that EGF stimulated HUVEC (human umbilical vein endothelial cells) proliferation in a concentration‐dependent manner, of which the maximum effective concentration of EGF was 10 ng/ml. Western blot analysis showed that EGF at 10 ng/ml significantly induced the phosphorylation of ERK1/2 (extracellular signal‐regulated kinase 1 and 2) and p38 kinase at 5 min, while it induced the phosphorylation of JNK/SAPK (c‐Jun N‐terminal kinase/stress‐activated protein kinase) at 15 min. Further results showed that a JNK/SAPK inhibitor, SP600125, and a specific siRNA JNK/SAPK could both significantly inhibit EGF‐induced tube formation in HUVEC cells, and an ERK1/2 inhibitor PD098059 could also block the tube formation in some content, while a p38 inhibitor SB203580 failed to do so. Furthermore, only SP600125 significantly inhibited EGF‐induced HUVEC cell proliferation under no cytotoxic concentration, so did JNK/SAPK siRNA. In conclusion, JNK/SAPK and ERK1/2 signals therefore play critical roles in EGF‐mediated HUVEC cell angiogenesis.     

ISG15 modification of filamin B negatively regulates the type I interferon‐induced JNK signalling pathway

Interferon (IFN)‐induced signalling pathways have essential functions in innate immune responses. In response to type I IFNs, filamin B tethers RAC1 and a Jun N‐terminal kinase (JNK)‐specific mitogen‐activated protein kinase (MAPK) module—MEKK1, MKK4 and JNK—and thereby promotes the activation of JNK and JNK‐mediated apoptosis. Here, we show that type I IFNs induce the conjugation of filamin B by interferon‐stimulated gene 15 (ISG15). ISGylation of filamin B led to the release of RAC1, MEKK1 and MKK4 from the scaffold protein and thus to the prevention of sequential activation of the JNK cascade. By contrast, blockade of filamin B ISGylation by substitution of Lys 2467 with arginine or by knockdown of ubiquitin‐activating enzyme E1‐like (UBEL1) prevented the release of the signalling molecules from filamin B, resulting in persistent promotion of JNK activation and JNK‐mediated apoptosis. These results indicate that filamin B ISGylation acts as a negative feedback regulatory gate for the desensitization of type I IFN‐induced JNK signalling.     

PGE2 inhibits MMP expression by suppressing MKK4–JNK MAP kinase–c‐JUN pathway via EP4 in human articular chondrocytes

Prostaglandin E2 (PGE2) is one of pro‐inflammatory mediators. PGE2 maintains the homeostasis of many organs including articular cartilage, and a previous report showed that continuous inhibition of PGE2 accelerates the progression of osteoarthritis (OA). While PGE2 inhibits matrix metalloprotease (MMP) expression in several types of cells, little is known on direct effects of PGE2 on MMP expression in articular chondrocytes. The objective of this study was to investigate direct effects of PGE2 on IL‐1β‐induced MMP‐1 and MMP‐13 expression and the intracellular signaling in articular chondrocytes. PGE2 showed inhibitory effects on IL‐1β‐induced MMP‐1 and MMP‐13 expression demonstrated by immunoblotting both in OA and normal chondrocytes, which was further confirmed by enzyme‐linked immunosorbent assay and immunohistochemistry of explant cultures of articular cartilages. An EP4 agonist, ONO‐AE1‐329, mimicked the inhibitory effect of PGE2, while an EP4 antagonist, ONO‐AE3‐208, blocked the effects. PGE2 suppressed the phosphorylation of JNK and ERK MAP kinases, but only knockdown of JNK by specific siRNA mimicked the effect of PGE2. PGE2 further inhibited the phosphorylation of MKK4 without suppression of MKK7 phosphorylation, and of c‐JUN to decrease expression levels of MMP‐1 and MMP‐13. These results demonstrate that PGE2 inhibits IL‐1β‐induced MMP‐1 and MMP‐13 productions via EP4 by suppressing MKK4–JNK MAP kinase–c‐JUN pathway. J. Cell. Biochem. 109: 425–433, 2010. © 2009 Wiley‐Liss, Inc.     

Involvement of the JNK activation in terbinafine‐induced p21 up‐regulation and DNA synthesis inhibition in human vascular endothelial cells

Previously, we demonstrated that the extracellular signal‐regulated kinase (ERK)‐mediated pathway contributes to the terbinafine (TB)‐induced increases of p21 and p53 protein level as well as decrease of DNA synthesis in human umbilical venous endothelial cells (HUVEC). The aim of this study is to examine the involvement of c‐Jun NH2‐terminal kinase (JNK) in the TB‐induced increase of p21 protein level and DNA synthesis inhibition. Western blot analysis and kinase assay demonstrated that TB treatment increased both the protein level and the kinase activity of JNK1/2 in HUVEC. Transfection of HUVEC with JNK1 dominant negative (DN‐JNK1) prevented the TB‐induced increases of p21 and p53 protein level and decrease of DNA synthesis, suggesting that JNK1/2 activation is involved in the TB‐induced cell cycle arrest in HUVEC. Moreover, over‐expression of mitogen‐activated protein kinase (MEK)‐1 prevented the TB‐induced increase of JNK1/2 protein levels, suggesting that MEK‐1 is an upstream inhibitor of JNK. Transfection of HUVEC with DN‐JNK1 prevented the TB‐induced inhibition of ERK phosphorylation, suggesting that JNK1/2 might serve as a negative regulator of ERK. Taken together, our results suggest that JNK activation is involved in the TB‐induced inhibition of ERK phosphorylation, p53 and p21 up‐regulation and DNA synthesis inhibition in HUVEC. J. Cell. Biochem. 108: 860–866, 2009. © 2009 Wiley‐Liss, Inc.     

14‐3‐3 Ligand Prevents Nuclear Import of c‐ABL Protein in Chronic Myeloid Leukemia

Here we demonstrated that the ‘loss of function’ of not‐rearranged c‐ABL in chronic myeloid leukemia (CML) is promoted by its cytoplasmic compartmentalization bound to 14‐3‐3 sigma scaffolding protein. In particular, constitutive tyrosine kinase (TK) activity of p210 BCR‐ABL blocks c‐Jun N‐terminal kinase (JNK) phosphorylation leading to 14‐3‐3 sigma phosphorylation at a critical residue (Ser¹⁸⁶) for c‐ABL binding in response to DNA damage. Moreover, it is associated with 14‐3‐3 sigma over‐expression arising from epigenetic mechanisms (promoter hyper‐acetylation). Accordingly, p210 BCR‐ABL TK inhibition by the TK inhibitor Imatinib mesylate (IM) evokes multiple events, including JNK phosphorylation at Thr¹⁸³, p38 mitogen‐activated protein kinase (MAPK) phosphorylation at Thr¹⁸⁰, c‐ABL de‐phosphorylation at Ser residues involved in 14‐3‐3 binding and reduction of 14‐3‐3 sigma expression, that let c‐ABL release from 14‐3‐3 sigma and nuclear import, and address BCR‐ABL‐expressing cells towards apoptotic death. Informational spectrum method (ISM), a virtual spectroscopy method for analysis of protein interactions based on their structure, and mathematical filtering in cross spectrum (CS) analysis identified 14‐3‐3 sigma/c‐ABL binding sites. Further investigation on CS profiles of c‐ABL‐ and p210 BCR‐ABL‐containing complexes revealed the mechanism likely involved 14‐3‐3 precluded phosphorylation in CML cells.     

Proteomic analysis of SP600125‐controlled TrkA‐dependent targets in SK‐N‐MC neuroblastoma cells: Inhibition of TrkA activity by SP600125

The c‐Jun N‐terminal kinase (JNK) is well known to play an important role in cell death signaling of the p75 neurotrophin receptor. However, little has been studied about a role of JNK in the signaling pathways of the tropomyosin‐related kinase A (TrkA) neurotrophin receptor. In this study, we investigated JNK inhibitor SP600125‐controlled TrkA‐dependent targets by proteomic analysis to better understand an involvement of JNK in TrkA‐mediated signaling pathways. PDQuest image analysis and protein identification results showed that hnRNP C1/C2, α‐tubulin, β‐tubulin homolog, actin homolog, and eIF‐5A‐1 protein spots were upregulated by ectopic expression of TrkA, whereas α‐enolase, peroxiredoxin‐6, PROS‐27, HSP70, PP1‐gamma, and PDH E1‐alpha were downregulated by TrkA, and these TrkA‐dependent upregulation and downregulation were significantly suppressed by SP600125. Notably, TrkA largely affected certain PTM(s) but not total protein amounts of the SP600125‐controlled TrkA‐dependent targets. Moreover, SP600125 strongly suppressed TrkA‐mediated tyrosine phosphorylation signaling pathways as well as JNK signaling, indicating that SP600125 could function as a TrkA inhibitor. Taken together, our results suggest that TrkA could play an important role in the cytoskeleton, cell death, cellular processing, and glucose metabolism through activation or inactivation of the SP600125‐controlled TrkA‐dependent targets.