Arsenic carcinogenicity: Relevance of c-Src activation

Environmental and occupational exposure to arsenic is associated with increased risk of skin, urinary bladder and respiratory tract cancers. Increasing evidence indicates that arsenic acts at the level of tumor promotion by modulating the signaling pathways responsible for cell growth. One of this pathways might include c-Src dependent EGFR and MAPK activation.     

Paradoxical activation of c-Src as a drug-resistant mechanism

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Functional role of c-Src in IL-1-induced NF-kappa B activation: c-Src is a component of the IKK complex

Interleukin-1 (IL-1) mediates numerous host responses through the rapid activation of nuclear factor-kappa B (NF-kappa B), but the signal pathways leading to NF-kappa B activation are regulated at multiple stages. Here, we propose a novel regulatory system for IL-1-induced NF-kappa B activation by a tyrosine kinase, c-Src. The kinase activity of c-Src increases in an IL-1-dependent manner and the ectopic expression of c-Src augments IL-1-induced NF-kappa B activation, suggesting the involvement of c-Src in IL-1 signaling. However, a Src family inhibitor, PP2 failed to inhibit IL-1-induced NF-kappa B activation, and the expression of a c-Src mutant lacking kinase activity (c-Src KD) augmented IL-1-induced NF-kappa B activation as well as wild type c-Src, indicating that the tyrosine kinase activity is not required for IL-1-induced NF-kappa B activation. Furthermore, a physiological interaction between c-Src and I kappa B kinase gamma (IKK gamma) was observed, implying the involvement of c-Src in the IKK-complex. While c-Src augmented IL-1-induced IKK activation independent of its kinase activity, the region comprising amino acids 361-440 in the c-Src kinase domain are required for NF-kappa B activation. The same region of c-Src is also required for IL-1-induced IKK activation and the association with IKK gamma. Taken together, our results suggest that c-Src plays a critical role in IL-1-induced NF-kappa B activation through the IKK complex.     

c-Src activation through a TrkA and c-Src interaction is essential for cell proliferation and hematological malignancies

Although the kinase receptor TrkA may play an important role in acute myeloid leukemia (AML), its involvement in other types of leukemia has not been reported. Furthermore, how it contributes to leukemogenesis is unknown. Here, we describe a molecular network that is important for TrkA function in leukemogenesis. We found that TrkA is frequently overexpressed in other types of leukemia such as acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), and myelodysplastic syndrome (MDS) including AML. In addition, TrkA was overexpressed in patients with MDS or secondary AML evolving from MDS. TrkA induced significant hematological malignancies by inducing PLK-1 and Twist-1, and enhanced survival and proliferation of leukemia, which was correlated with activation of the phosphatidylinositol 3-kinase/Akt/mTOR pathway. Moreover, endogenous TrkA associated with c-Src complexes was detected in leukemia. Suppression of c-Src activation by TrkA resulted in markedly decreased expression of PLK-1 and Twist-1 via suppressed activation of Akt/mTOR cascades. These data suggest that TrkA plays a key role in leukemogenesis and reveal an unexpected physiological role for TrkA in the pathogenesis of leukemia. These data have important implications for understanding various hematological malignancies.     

The crystal structure of a c-Src complex in an active conformation suggests possible steps in c-Src activation

The regulation of the activity of Abl and Src family tyrosine kinases is mediated by intramolecular interactions between the SH3, SH2, and kinase (SH1) domains. We have determined the crystal structure of an unphosphorylated form of c-Src in which the SH2 domain is not bound to the C-terminal tail. This results in an open structure where the kinase domain adopts an active conformation and the C terminus binds within a hydrophobic pocket in the C-terminal lobe. NMR binding studies support the hypothesis that an N-terminal myristate could bind in this pocket, as observed for Abl, suggesting that c-Src may also be regulated by myristate binding. In addition, the structure contains a des-methyl analog of the antileukemia drug imatinib (STI571; Gleevec). This structure reveals why the drug shows a low affinity for active kinase conformations, contributing to its excellent kinase selectivity profile.     

S-nitrosylation of c-Src via NMDAR-nNOS module promotes c-Src activation and NR2A phosphorylation in cerebral ischemia/reperfusion

Previous studies suggested that activated c-Src promote the tyrosine phosphorylation of NMDA receptor subunit NR2A, and thus aggravate the injury induced by transient cerebral ischemia/reperfusion (I/R) in rat hippocampus CA1 region. In this study, we examined the effect of nitric oxide (NO) on the activation of c-Src and the tyrosine phosphorylation of NMDA receptor NR2A subunit. The results show that S-nitrosylation and the phosphorylation of c-Src were induced after cerebral I/R in rats, and administration of nNOS inhibitor 7-NI, nNOS antisense oligonucleotides and exogenous NO donor sodium nitroprusside diminished the increased S-nitrosylation and phosphorylation of c-Src during cerebral I/R. The cysteine residues of c-Src modified by S-nitrosylation are Cys489, Cys498, and Cys500. On the other hand, NMDAR antagonist MK-801 could attenuate the S-nitrosylation and activation of c-Src. Taken together, the S-nitrosylation of c-Src is provoked by NO derived from endogenous nNOS, which is activated by Ca²⁺ influx from NMDA receptors, and promotes the auto-phosphorylation at tyrosines and further phosphorylates NR2A. The molecular mechanism we outlined here is a novel postsynaptic NMDAR-nNOS/c-Src-mediated signaling amplification, the ‘NMDAR-nNOS → NO → SNO-c-Src → p-c-Src → NMDAR-nNOS’ cycle, which presents the possibility as a potential therapeutic target for stroke treatment.     

Endosomal NADPH oxidase regulates c-Src activation following hypoxia/reoxygenation injury

c-Src has been shown to activate NF-kappaB (nuclear factor kappaB) following H/R (hypoxia/reoxygenation) by acting as a redox-dependent IkappaBalpha (inhibitory kappaB) tyrosine kinase. In the present study, we have investigated the redox-dependent mechanism of c-Src activation following H/R injury and found that ROS (reactive oxygen species) generated by endosomal Noxs (NADPH oxidases) are critical for this process. Endocytosis following H/R was required for the activation of endosomal Noxs, c-Src activation, and the ability of c-Src to tyrosine-phosphorylate IkappaBalpha. Quenching intra-endosomal ROS during reoxygenation inhibited c-Src activation without affecting c-Src recruitment from the plasma membrane to endosomes. However, siRNA (small interfering RNA)-mediated knockdown of Rac1 prevented c-Src recruitment into the endosomal compartment following H/R. Given that Rac1 is a known activator of Nox1 and Nox2, we investigated whether these two proteins were required for c-Src activation in Nox-deficient primary fibroblasts. Findings from these studies suggest that both Nox1 and Nox2 participate in the initial redox activation of c-Src following H/R. In summary, our results suggest that Rac1-dependent Noxs play a critical role in activating c-Src following H/R injury. This signalling pathway may be a useful therapeutic target for ischaemia/reperfusion-related diseases.     

Differential activation and redistribution of c-Src and Fyn in platelets, assessed by MoAb specific for C-terminal tyrosine-dephosphorylated c-Src and Fyn

Tyrosine kinases, c-Src and Fyn, in their active form, have their C-terminal tyrosine residue dephosphorylated. In this study, we used clone 28, a monoclonal antibody (MoAb) that recognizes dephosphorylated C-terminal tyrosine of c-Src and Fyn, to investigate the mode of activation and mobilization of these kinases. Independently of integrin alphaIIbbeta3 signaling, the Fyn activity increased by 8.3-fold 5 s after stimulation with 20 microM TRAP (thrombin receptor agonist peptide), while that of c-Src increased only by 2.9-fold 15 s after stimulation. Both c-Src and Fyn translocated to the Triton-insoluble cytoskeletal fraction in an aggregation-dependent manner. Five minutes after TRAP-stimulation, 85% of Fyn translocated to the cytoskeleton, while only about 20% of c-Src was recovered in this fraction. The Triton-insoluble fraction was further fractionated by RIPA (radioimmunoprecipitation assay) buffer containing 0.1% SDS. While active c-Src was predominantly present in the Triton-insoluble/RIPA-insoluble fraction, clone 28-negative c-Src was present in the Triton-insoluble/RIPA-soluble fraction. On the other hand, Fyn was present only in the Triton-insoluble/RIPA-insoluble fraction. These findings suggest that the mode of activation and redistribution into the cytoskeleton differs between c-Src and Fyn, and that clone 28 provides a useful tool for investigating the activation and mobilization of Src family tyrosine kinases.     

Suppression of c-Src activity stimulates muscle differentiation via p38 MAPK activation

Role of c-Src in muscle differentiation has been controversial. Here, we investigated if c-Src positively or negatively regulates muscle differentiation, using H9c2 and C2C12 cell lines. Inhibition of c-Src by treatment with PP1 and SU6656, pharmacologic inhibitors of Src family kinases, or by expression of a dominant negative c-Src, all induced muscle differentiation in proliferation medium (PM). In differentiating cells in differentiation medium (DM), c-Src activity gradually decreased and reached basal level 3 days after induction of differentiation. Inhibition of c-Src suppressed Raf/MEK/ERK pathway but activated p38 MAPK. Inhibition of p38 MAPK did not affect c-Src activity in PM. However, it reactivated Raf/MEK/ERK pathway in c-Src-inhibited cells regardless of PM or DM. Concomitant inhibition of c-Src and p38 MAPK activities blocked muscle differentiation in both media. In conclusion, suppression of c-Src activity stimulates muscle differentiation by activating p38 MAPK uni-directionally.     

HBx protein induces EMT through c-Src activation in SMMC-7721 hepatoma cell line

The relationships between epithelial-to-mesenchymal transition (EMT), hepatitis B virus X protein (HBx), and the non-receptor tyrosine kinase c-Src were investigated. The HBx gene transfected SMMC-7721 cells underwent morphological changes from a classic epithelial morphology to a spindle-like shape. The HBx transfection increased the invasive potential of these cells. When the transfected cells were exposed to the c-Src kinase inhibitor PP2, the cells recovered their original epithelial morphology and the mRNA and protein expression of epithelial and mesenchymal markers returned to the parental cell levels. Our data suggest that activated c-Src played a critical role in the HBx-induced EMT of SMMC-7721 cells.     

Up-regulation of AMP-activated protein kinase in cancer cell lines is mediated through c-Src activation

We report that the activation level of AMP-dependent protein kinase AMPK is elevated in cancer cell lines as a hallmark of their transformed state. In OVCAR3 and A431 cells, c-Src signals through protein kinase Cα, phospholipase Cγ, and LKB1 to AMPK. AMPK controls internal ribosome entry site (IRES) dependent translation in these cells. We suggest that AMPK activation via PKC might be a general mechanism to regulate IRES-dependent translation in cancer cells.     

Differential mitotic activation of endogenous c-Src, c-Yes, and Lyn in HeLa cells

Src-family tyrosine kinases (SFKs) play an important role in mitosis. Despite overlapping expression of multiple SFK members, little is known about how individual SFK members are activated in M phase. Here, we examined mitotic activation of endogenous c-Src, c-Yes, and Lyn, which are co-expressed in HeLa cells. c-Src, c-Yes, and Lyn were activated at different levels in M phase, and the activation was inhibited by Cdc2 inactivation. Mitotic c-Src and c-Yes exhibited normal- and retarded-electrophoretic-mobility forms on SDS-polyacrylamide gels, whereas Lyn did not show mobility retardation. Like c-Src, the retardation of electrophoretic mobility of c-Yes was caused by Cdc2-mediated phosphorylation. The normal- and retarded-mobility forms of c-Src were comparably activated, but activation of the retarded-mobility form of c-Yes was higher than that of the normal-mobility form of c-Yes. Thus, these results suggest that endogenous c-Src, c-Yes, and Lyn are differentially activated through Cdc2 activation during M phase.     

Saturated fatty acids induce c-Src clustering within membrane subdomains, leading to JNK activation

Saturated fatty acids (FA) exert adverse health effects and are more likely to cause insulin resistance and type 2 diabetes than unsaturated FA, some of which exert protective and beneficial effects. Saturated FA, but not unsaturated FA, activate Jun N-terminal kinase (JNK), which has been linked to obesity and insulin resistance in mice and humans. However, it is unknown how saturated and unsaturated FA are discriminated. We now demonstrate that saturated FA activate JNK and inhibit insulin signaling through c-Src activation. FA alter the membrane distribution of c-Src, causing it to partition into intracellular membrane subdomains, where it likely becomes activated. Conversely, unsaturated FA with known beneficial effects on glucose metabolism prevent c-Src membrane partitioning and activation, which are dependent on its myristoylation, and block JNK activation. Consumption of a diabetogenic high-fat diet causes the partitioning and activation of c-Src within detergent insoluble membrane subdomains of murine adipocytes.     

Beryllium: genotoxicity and carcinogenicity

Beryllium (Be) has physical-chemical properties, including low density and high tensile strength, which make it useful in the manufacture of products ranging from space shuttles to golf clubs. Despite its utility, a number of standard setting agencies have determined that beryllium is a carcinogen. Only a limited number of studies, however, have addressed the underlying mechanisms of the carcinogenicity and mutagenicity of beryllium. Importantly, mutation and chromosomal aberration assays have yielded somewhat contradictory results for beryllium compounds and whereas bacterial tests were largely negative, mammalian test systems showed evidence of beryllium-induced mutations, chromosomal aberrations, and cell transformation. Although inter-laboratory differences may play a role in the variability observed in genotoxicity assays, it is more likely that the different chemical forms of beryllium have a significant effect on mutagenicity and carcinogenicity. Because workers are predominantly exposed to airborne particles which are generated during the machining of beryllium metal, ceramics, or alloys, testing of the mechanisms of the mutagenic and carcinogenic activity of beryllium should be performed with relevant chemical forms of beryllium.