PTPIP51, a novel 14-3-3 binding protein, regulates cell morphology and motility via Raf-ERK pathway

Cell migration plays a critical role during the development of most organisms and the process of malignant tumor metastasis. In the present study, we investigated the role of PTPIP51 (protein tyrosine phosphatase interacting protein 51) in cell motility. Overexpression of PTPIP51 induced cell elongation, increased cell migration, adhesion, and spreading, while downregulation of PTPIP51 had the opposite effects. We demonstrated here, that PTPIP51 could regulate ERK activity on Raf level, since MEK inhibitor and dominant-negative Raf-1 but not Ras could inhibit the ERK activation induced by PTPIP51. Further studies proved that PTPIP51 could interact with Raf-1 through 14–3–3, suggesting that PTPIP51 is a regulator of the Raf–MEK–ERK cascade through modulation of Raf-1 by 14–3–3. In addition, two redundant 14–3–3 binding domains in the PTPIP51 protein have been identified by deletion/mutation studies. We conclude that PTPIP51 regulates cell morphology and cell motility via interaction with Raf-1 through 14–3–3, and that PTPIP51 binds to 14–3–3 through two redundant binding domains.     

Peroxynitrite activates mitogen-activated protein kinase (MAPK) via a MEK-independent pathway: a role for protein kinase C.

In this study we show that phosphorylation of extracellular signal-regulated kinase (ERK1/2; also known as p44/42MAPK) following peroxynitrite (ONOO(-)) exposure occurs via a MAPK kinase (MEK)-independent but PKC-dependent pathway in rat-1 fibroblasts. ONOO(-)-mediated ERK1/2 phosphorylation was not blocked by MEK inhibitors PD98059 and U0126. Furthermore, no increase in MEK phosphorylation was detected upon ONOO(-) treatment. Staurosporine was used to investigate whether protein kinase C (PKC) is involved. This was confirmed by down-regulation of PKC by phorbol-12,13-dibutyrate, which resulted in significant reduction of ERK1/2 phosphorylation by ONOO(-), implying that activation of ERK by ONOO(-) depends on activation of PKC. Indeed, PKCalpha and epsilon were activated upon ONOO(-) exposure. When cells were treated with ONOO(-) in a calcium-free buffer, no activation of PKCalpha was detected. Concomitantly, a reduction of ERK1/2 phosphorylation was observed suggesting that calcium was required for translocation of PKCalpha and ERK phosphorylation by ONOO(-). Indeed, ONOO(-) exposure resulted in increased cytosolic calcium, which depended on the presence of extracellular calcium. Finally, data using G?6976, an inhibitor of calcium-dependent PKC activation, implied that ONOO(-)-mediated ERK1/2 phosphorylation depends on activation of a calcium-dependent PKC.     

MAPK signaling to the early secretory pathway revealed by kinase/phosphatase functional screening

To what extent the secretory pathway is regulated by cellular signaling is unknown. In this study, we used RNA interference to explore the function of human kinases and phosphatases in controlling the organization of and trafficking within the secretory pathway. We identified 122 kinases/phosphatases that affect endoplasmic reticulum (ER) export, ER exit sites (ERESs), and/or the Golgi apparatus. Numerous kinases/phosphatases regulate the number of ERESs and ER to Golgi protein trafficking. Among the pathways identified, the Raf–MEK (MAPK/ERK [extracellular signal-regulated kinase] kinase)–ERK cascade, including its regulatory proteins CNK1 (connector enhancer of the kinase suppressor of Ras-1) and neurofibromin, controls the number of ERESs via ERK2, which targets Sec16, a key regulator of ERESs and COPII (coat protein II) vesicle biogenesis. Our analysis reveals an unanticipated complexity of kinase/phosphatase-mediated regulation of the secretory pathway, uncovering a link between growth factor signaling and ER export.     

Inflammation and MiR-21 Pathways Functionally Interact to Downregulate PDCD4 in Colorectal Cancer

Inflammation plays a direct role in colorectal cancer (CRC) progression; however the molecular mechanisms responsible for this effect are unclear. The inflammation induced cyclooxygenase 2 (COX-2) enzyme required for the production of Prostaglandin E2 (PGE₂), can promote colorectal cancer by decreasing expression of the tumour suppressor gene Programmed Cell Death 4 (PDCD4). As PDCD4 is also a direct target of the oncogene microRNA-21 (miR-21) we investigated the relationship between the COX-2 and miR-21 pathways in colorectal cancer progression. Gene expression profile in tumour and paired normal mucosa from 45 CRC patients demonstrated that up-regulation of COX-2 and miR-21 in tumour tissue correlates with worse Dukes'' stage. In vitro studies in colonic adenocarcinoma cells revealed that treatment with the selective COX-2 inhibitor NS398 significantly decreased miR-21 levels (p = 0.0067) and increased PDCD4 protein levels (p<0.001), whilst treatment with PGE₂ up-regulated miR-21 expression (p = 0.019) and down-regulated PDCD4 protein (p<0.05). These findings indicate that miR-21 is a component of the COX-2 inflammation pathway and that this pathway promotes worsening of disease stage in colorectal cancer by inducing accumulation of PGE₂ and increasing expression of miR-21 with consequent downregulation of the tumour suppressor gene PDCD4.     

Oligomeric aggregates of amyloid β peptide 1–42 activate ERK/MAPK in SH-SY5Y cells via the α7 nicotinic receptor

The production and aggregation of amyloid β peptides (Aβ) has been linked to the development and progression of Alzheimer's disease. It is apparent that the various structural forms of Aβ can affect cell signalling pathways and the activity of neurons differently. In this study, we investigated the effects of oligomeric and fibrillar aggregates of Aβ 1–42 (Aβ42) and non-aggregated peptide upon activation of the ERK/MAPK signalling pathway. In SH-SY5Y cells, acute exposure to oligomeric Aβ42 led to phosphorylation of ERK1/2 at concentrations as low as 1 nM and up to 100 nM. These changes were detected as early as 5 min following exposure to 100 nM oligomeric Aβ42, reaching a maximum level after 10 min. Phosphorylation of ERK1/2 subsequently declined to and remained at basal levels after 30 min to 2 h of exposure. Fibrillar aggregates of Aβ42 did not significantly induce phosphorylation of ERK1/2 and non-aggregated Aβ42 did not activate the pathway. The effects of oligomeric Aβ42 to increase ERK phosphorylation above basal levels were inhibited by MLA, a specific antagonist of the α7 nAChR. U0126, an inhibitor of MEK, the upstream activator of ERK1/2, completely blocked induction of ERK1/2 phosphorylation. Oligomeric aggregates of Aβ42 are the principal structural form of the peptide that activates ERK/MAPK in SH-SY5Y cells and these effects are mediated by the α7 nAChR.     

High‐fat diet‐induced dysbiosis mediates MCP‐1/CCR2 axis‐dependent M2 macrophage polarization and promotes intestinal adenoma‐adenocarcinoma sequence

High‐fat diet (HFD) is a well‐known risk factor for gut microbiota dysbiosis and colorectal cancer (CRC). However, evidence relating HFD, gut microbiota and carcinogenesis is limited. Our study aimed to demonstrate that HFD‐induced gut dysbiosis promoted intestinal adenoma‐adenocarcinoma sequence. In clinical study, we found that HFD increased the incidence of advanced colorectal neoplasia (AN). The expression of monocyte chemoattractant protein 1 (MCP‐1), CC chemokine receptor 2 (CCR2) and CD163 in CRC patients with HFD was significantly higher than that in CRC patients with normal diet. When it comes to the Apc^min/+ mice, HFD consumption could induce gut dysbiosis and promote intestinal carcinogenesis, accompanying with activation of MCP‐1/CCR2 axis that recruited and polarized M2 tumour‐associated macrophages. Interestingly, transfer of faecal microbiota from HFD‐fed mice to another batch of Apc^min/+ mice in the absence of HFD could also enhance carcinogenesis without significant body weight gain and induced MCP‐1/CCR2 axis activation. HFD‐induced dysbiosis could also be transmitted. Meanwhile, antibiotics cocktail treatment was sufficient to inhibit HFD‐induced carcinogenesis, indicating the vital role of dysbiosis in cancer development. Conclusively, these data indicated that HFD‐induced dysbiosis accelerated intestinal adenoma‐adenocarcinoma sequence through activation of MCP‐1/CCR2 axis, which would provide new insight into better understanding of the mechanisms and prevention for HFD‐related CRC.     

ZC3H13 suppresses colorectal cancer proliferation and invasion via inactivating Ras–ERK signaling

ZC3H13 is a canonical CCCH zinc finger protein, which harbors a somatic frame-shift mutation in colorectal cancer (CRC). However, its expression and biological function were still uncertain. In the current study, we found that ZC3H13 was served as a tumor suppressor in CRC cells, which decreased the expression of Snail, Cyclin D1, and Cyclin E1, and increased the expression of Occludin and Zo-1 through inactivating Ras–ERK signaling pathway. Furthermore, reduction of ZC3H13 associated with advanced TNM stage (p = 0.02), positive regional lymph node metastasis ( p = 0.01). Taken together, the current study indicated that ZC3H13 may be an upstream regulator of Ras–ERK signaling pathway and suppressed invasion and proliferation of CRC.     

Regulation of the Ras‐GTPase activating protein neurofibromin by C‐tail phosphorylation: implications for protein kinase C/Ras/extracellular signal‐regulated kinase 1/2 pathway signaling and neuronal differentiation

PKC, Ras, and ERK1/2 signaling is pivotal to differentiation along the neuronal cell lineage. One crucial protein that may play a central role in this signaling pathway is the Ras GTPase‐activating protein, neurofibromin, a PKC substrate that may exert a positive role in neuronal differentiation. In this report, we studied the dynamics of PKC/Ras/ERK pathway signaling, during differentiation of SH‐SY5Y neuroblastoma cells upon treatment with the PKC agonist, phorbol ester 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA). Surprisingly, we observed that, among other PKC‐dependent signaling events, TPA induced a rapid and sustained decrease of neurofibromin immunoreactivity which was not due to proteolysis. Instead, we identified a specific phosphorylation event at the C‐tail of neurofibromin. This phosphorylation was acute and correlated perfectly with the signaling dynamics of the Ras/ERK pathway. Moreover, it persisted throughout prolonged treatment and TPA‐induced differentiation of SH‐SY5Y cells, concurrently with sustained activation of ERK1/2. Most importantly, C‐tail phosphorylation of neurofibromin correlated with a shift of neurofibromin localization from the nucleus to the cytosol. We propose that PKC‐dependent, sustained C‐tail phosphorylation is a requirement for prolonged recruitment of neurofibromin from the nucleus to the cytosol in order for a fine regulation of Ras/ERK pathway activity to be achieved during differentiation.     

OX40–OX40 ligand interaction may activate phospholipase C signal transduction pathway in human umbilical vein endothelial cells

Studies have recently supported the emerging role of OX40/OX40L interaction in atherosclerosis. The mechanism of OX40/OX40L interaction may be related to a variety of signal pathways. The most important signal pathway involves the activation of phospholipase C (PLC) which induces diacylglycerol–protein kinase C (DAG–PKC) and the inositol trisphosphate (IP₃)–intracellular free calcium ([Ca²⁺]_i) pathway. The aim of this work was to investigate whether OX40–OX40L interaction can stimulate the PLC signal pathway in human umbilical vein endothelial cells (HUVEC). The DAG and IP₃ level in HUVEC were measured by radio-enzymatic assay. The activity of PKC was detected by its ability to transfer phosphate from [γ-³²P]ATP to lysine-rich histone. [Ca²⁺]_i concentrations were measured by flow cytometric analysis. Results showed that the DAG level was markedly increased in a concentration-dependent, biphasic manner in HUVEC induced by OX40. The early phase was rapid, peaking at 30 s. The late phase reached the maximum level at 15 min and decayed slowly. OX40 increased PKC activity in a dose-dependent manner with two peaks at 40–50 s and 12–16 min, then decreased slowly, yet maintained a high level for at least 30 min. PKC activity was mainly in cytosol at rest and translocated from cytosol to membrane when stimulated by OX40. Similarly, OX40-induced rapid IP₃ formation coincided with the peak of DAG level. Moreover, OX40 also induced peak [Ca²⁺]_i responses including the rapid transient phase and the sustained phase. Anti-OX40L antibody significantly suppressed OX40-induced DAG–PKC and IP₃–[Ca²⁺]_i signal pathway activation in HUVEC. In conclusion, the data suggested that OX40–OX40L interaction induced a robust stimulation of phospholipase C signal transduction pathway in HUVEC.     

Phosphorylation of Raf-1 by Kinase Suppressor of Ras Is Inhibited by “MEK-Specific” Inhibitors PD 098059 and U0126 in Differentiating HL60 Cells

Determination of the involvement of MAP kinase cascades in signaling cell growth or differentiation is aided by the use of the inhibitors PD 098059 [2-(2′-amino-3′-methoxyphenyl)oxananphthalen-4-one] and U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene], believed to be MEK-specific kinase inhibitors. We report here that the activity of kinase suppressor of ras (KSR-1), a kinase upstream of raf-1, is inhibited by both these compounds at concentrations similar to those that inhibit MEK-1. Further, in HL60 cells induced to differentiate with 1,25-dihydroxyvitamin D₃ raf-1 and p90RSK, but not ERK1/2, are coregulated, and their expression as well as monocytic differentiation is inhibited in parallel by PD 098059. Thus, in this system raf-1 is phosphorylated by KSR-1, and PD 098059 as well as U0126 inhibits this phosphorylation. This suggests great caution in the interpretation of experiments that utilize these pharmacological inhibitors of kinase activity as evidence for a role for the MEK–ERK module in ras or raf-1 signaling.     

Role of PKC and MAPK in cytosolic PLA2 phosphorylation and arachadonic acid release in primary murine astrocytes

Although Group IV cytosolic phospholipase A2 (cPLA2) in astrocytes has been implicated in a number of neurodegenerative diseases, mechanisms leading to its activation and release of arachidonic acid (AA) have not been clearly elucidated. In primary murine astrocytes, phorbol myristate acetate (PMA) and ATP stimulated phosphorylation of ERK1/2 and cPLA2 as well as evoked AA release. However, complete inhibition of phospho-ERK by U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK), did not completely inhibit PMA-stimulated cPLA2 and AA release. Epidermal growth factor (EGF) also stimulated phosphorylation of ERK1/2 and cPLA2[largely through a protein kinase C (PKC)-independent pathway], but EGF did not evoke AA release. These results suggest that phosphorylation of cPLA2 due to phospho-ERK is not sufficient to evoke AA release. However, complete inhibition of ATP-induced cPLA2 phosphorylation and AA release was observed when astrocytes were treated with GF109203x, a general PKC inhibitor, together with U0126, indicating the important role for both PKC and ERK in mediating the ATP-induced AA response. There is evidence that PMA and ATP stimulated AA release through different PKC isoforms in astrocytes. In agreement with the sensitivity of PMA-induced responses to PKC down-regulation, prolonged treatment with PMA resulted in down-regulation of PKCalpha and epsilon in these cells. Furthermore, PMA but not ATP stimulated rapid translocation of PKCalpha from cytosol to membranes. Together, our results provided evidence for an important role of PKC in mediating cPLA2 phosphorylation and AA release in astrocytes through both ERK1/2-dependent and ERK1/2-independent pathways.     

Mutant K-Ras activation of the proapoptotic MST2 pathway is antagonized by wild-type K-Ras

K-Ras mutations are frequent in colorectal cancer (CRC), albeit K-Ras is the only Ras isoform that can elicit apoptosis. Here, we show that mutant K-Ras directly binds to the tumor suppressor RASSF1A to activate the apoptotic MST2-LATS1 pathway. In this pathway LATS1 binds to and sequesters the ubiquitin ligase Mdm2 causing stabilization of the tumor suppressor p53 and apoptosis. However, mutant Ras also stimulates autocrine activation of the EGF receptor (EGFR) which counteracts mutant K-Ras-induced apoptosis. Interestingly, this protection requires the wild-type K-Ras allele, which inhibits the MST2 pathway in part via AKT activation. Confirming the pathophysiological relevance of the molecular findings, we find a negative correlation between K-Ras mutation and MST2 expression in human CRC patients and CRC mouse models. The small number of tumors with co-expression of mutant K-Ras and MST2 has elevated apoptosis rates. Thus, in CRC, mutant K-Ras transformation is supported by the wild-type allele.     

Developmental expression changes of the genes involved in IGFI signaling pathway in longissimus dorsi muscle of Tongcheng and Yorkshire pigs during postnatal growth

IGFI signaling pathway is sufficient to regulate myofibre hypertrophy postnatally, which is associated with muscle mass in economically livestock. In the present study, we drafted the developmental expression pattern of eight genes implicated in IGFI system across six stages of postnatal myofibre growth in Yorkshire and Tongcheng pigs. The results indicated that GRB2 may contribute to increased DNA content in postnatal myofibre hypertrophy via GRB2–Ras–Raf–MEK–ERK sub-pathway; INSR, PDK1, IRS1 and eIF4E may contribute to high growth rate via stimulating the rate of protein synthesis and inhibiting the rate of protein degradation. In addition, the results suggested 60 days maybe a very important stage in postnatal myofibre growth. Moreover, higher mRNA level of IRS1 and GLUT4 maybe associated with inferior meat quality in Yorkshire compared to Tongcheng pig. Therefore, IGFI signaling pathway regulates myofibre hypertrophy postnatally via complicated signal effectors, which may have negative impact on meat quality simultaneously.     

Involvement of Protein Kinase D1 in Signal Transduction from the Protein Kinase C Pathway to the Tyrosine Kinase Pathway in Response to Gonadotropin-releasing Hormone

The receptor for gonadotropin-releasing hormone (GnRH) belongs to the G protein-coupled receptors (GPCRs), and its stimulation activates extracellular signal-regulated protein kinase (ERK). We found that the transactivation of ErbB4 was involved in GnRH-induced ERK activation in immortalized GnRH neurons (GT1–7 cells). We found also that GnRH induced the cleavage of ErbB4. In the present study, we examined signal transduction for the activation of ERK and the cleavage of ErbB4 after GnRH treatment. Both ERK activation and ErbB4 cleavage were completely inhibited by YM-254890, an inhibitor of G_q/11 proteins. Down-regulation of protein kinase C (PKC) markedly decreased both ERK activation and ErbB4 cleavage. Experiments with two types of PKC inhibitors, Gö 6976 and bisindolylmaleimide I, indicated that novel PKC isoforms but not conventional PKC isoforms were involved in ERK activation and ErbB4 cleavage. Our experiments indicated that the novel PKC isoforms activated protein kinase D (PKD) after GnRH treatment. Knockdown and inhibitor experiments suggested that PKD1 stimulated the phosphorylation of Pyk2 by constitutively activated Src and Fyn for ERK activation. Taken together, it is highly possible that PKD1 plays a critical role in signal transduction from the PKC pathway to the tyrosine kinase pathway. Activation of the tyrosine kinase pathway may be involved in the progression of cancer.     

TUFT1 Facilitates Metastasis,Stemness, and Vincristine Resistance in Colorectal Cancer via Activation of PI3K/AKT Pathway

Since the incidence and mortality of colorectal cancer (CRC) are increasing in recent years, the research on the pathogenesis of colorectal cancer has attracted more and more attention. Here, our results confirmed that the mRNA expression level and proteins accumulation of TUFT1 were significantly increased in CRC tissues from late-stage CRC patients (III?+?IV) (p?<?0.001), indicated by qPCR and IHC assay. The TUFT1 expression was positively correlated with tumor stage by analyzing 126 specimens from CRC patients. Next, we found that up-regulation of TUFT1 enhanced the migration and invasion of LoVo cells, whereas the down-regulation of TUFT1 observably weakened the migration and invasion of SW837 cells, indicating that TUFT1 promotes the metastasis of CRC cells. In addition, TUFT1 overexpression increased the number of mammary spheres and vincristine resistance of LoVo cells by sphere formation assay and measuring the IC50 value, suggesting the TUFT1 promotes stemness and the vincristine resistance of CRC cells. Finally, we found that TUFT1 overexpression increased p-AKT in LoVo cells, while down-regulation of TUFT1 decreased the p-AKT levels in SW837 cells. Therefore, we determined that the function of TUFT1 in CRC depends on PI3K/AKT pathway. Taken together, these data demonstrated that TUFI1 facilitates metastasis, stemness, and vincristine resistance of colorectal cancer cells via activation of PI3K/AKT pathway, which might act as a promising therapeutic target for CRC.

     

Distinct Kinetic and Spatial Patterns of Protein Kinase C (PKC)- and Epidermal Growth Factor Receptor (EGFR)-dependent Activation of Extracellular Signal-regulated Kinases 1 and 2 by Human Nicotinic Acid Receptor GPR109A

Nicotinic acid (niacin) has been widely used as a lipid-lowering drug for several decades, and recently, orphan G protein-coupled receptor GPR109A has been identified as a receptor for niacin. Mechanistic investigations have shown that, upon niacin activation, GPR109A couples to a G_i protein and inhibits adenylate cyclase activity, leading to inhibition of liberation of free fatty acid. However, the underlying molecular mechanisms for GPR109A signaling remain largely unknown. Using CHO-K1 cells stably expressing GPR109A and A431 cells, which are a human epidermoid cell line with high levels of endogenous expression of functional GPR109A receptors, we found that activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by niacin was rapid, peaking at 5 min, and was significantly blocked by pertussis toxin. Furthermore, time course experiments with different kinase inhibitors demonstrated that GPR109A induced ERK1/2 activation via the matrix metalloproteinase/epidermal growth factor receptor transactivation pathway at both early and later time points (2–5 min); this pathway was distinct from the PKC pathway-mediated ERK1/2 phosphorylation that occurs at early time points (≤2 min) in response to niacin. Overexpression of Gβγ subunit scavengers βARK1-CT and the Gα subunit of transducin led to a significant reduction of ERK1/2 phosphorylation, suggesting a critical role for βγ subunits in GPR109A-activated ERK1/2 phosphorylation. Using arrestin-2/3-specific siRNA and an internalization-deficient GPR109A mutant, we found that arrestin-2 and arrestin-3 were not involved in GPR109A-mediated ERK1/2 activation. In conclusion, our findings demonstrate that upon binding to niacin GPR109A receptors initially activate G_i, leading to dissociation of the Gβγ subunit from activated G_i, and subsequently induce ERK1/2 activation via two distinct pathways, one PKC-dependent pathway occurring at a peak time of ≤2 min and the other matrix metalloproteinase-dependent growth factor receptor transactivation occurring at both early and later time points (2–5 min).