Sally A. Prigent

Relevance of mitogen activated protein kinase (MAPK) and phosphotidylinositol-3-kinase/protein kinase B (PI3K/PKB) pathways to induction of apoptosis by curcumin in breast cells.

Following observations that curcumin inhibited proliferation (IC(50)=1-5 microM), invasiveness and progression through S/G2/M phases of the cell cycle in the non-tumourigenic HBL100 and tumourigenic MDA-MB-468 human breast cell lines, it was noted that apoptosis was much more pronounced in the tumour line. Therefore, the ability of curcumin to modulate signalling pathways which might contribute to cell survival was investigated. After pre-treatment of cells for 20 min, curcumin (40 microM) inhibited EGF-stimulated phosphorylation of the EGFR in MDA-MB-468 cells and phosphorylation of extracellular signal regulated kinases (ERKs) 1 and 2, as well as ERK activity and levels of nuclear c-fos in both cell lines. At a lower dose (10 microM), it also inhibited the ability of anisomycin to activate JNK, resulting in decreased c-jun phosphorylation, although it did not inhibit JNK activity directly. In contrast, the activation of p38 mitogen activated protein kinase (MAPK) by anisomycin was not inhibited. Curcumin inhibited basal phosphorylation of Akt/protein kinase B (PKB) in both cell lines, but more consistently and to a greater extent in the MDA-MB-468 cells. The MAPK kinase (MKK) inhibitor U0126 (10 microM), while preventing ERK phosphorylation in MDA-MB-468 cells, did not induce apoptosis. The PI3K inhibitor LY294002 (50 microM) inhibited PKB phosphorylation in both cells lines, but only induced apoptosis in the MDA-MB-468 line. These results suggest that while curcumin has several different molecular targets within the MAPK and PI3K/PKB signalling pathways that could contribute to inhibition of proliferation and induction of apoptosis, inhibition of basal activity of Akt/PKB, but not ERK, may facilitate apoptosis in the tumour cell line.

The Shc-related adaptor protein, Sck, forms a complex with the vascular-endothelial-growth-factor receptor KDR in transfected cells

Despite much progress in recent years, the precise signalling events triggered by the vascular-endothelial-growth-factor (VEGF) receptors, fms-like tyrosine kinase (Flt1) and kinase insert domain-containing receptor (KDR), are incompletely defined. Results obtained when Flt1 and KDR are individually expressed in fibroblasts or porcine aortic endothelial cells have not been entirely consistent with those observed in other endothelial cells expressing both receptors endogenously. It has also been difficult to demonstrate VEGF-induced phosphorylation of Flt1, which has led to speculation that KDR may be the more important receptor for the mitogenic action of VEGF on endothelial cells. In an attempt to identify physiologically important effectors which bind to KDR, we have screened a yeast two-hybrid mouse embryo library with the cytoplasmic domain of KDR. Here we describe the identification of the adaptor protein, Shc-like protein (Sck), as a binding partner for KDR. We demonstrate that this interaction requires phosphorylation of KDR, and identify the binding site for the Src-homology 2 (SH2) domain as tyrosine-1175 of KDR. We have also shown that the SH2 domain of Sck, but not that of Src-homology collagen protein (Shc), can precipitate phosphorylated KDR from VEGF-stimulated porcine aortic endothelial cells expressing KDR, and that an N-terminally truncated Sck protein can associate with KDR, in a phosphorylation-dependent fashion, when co-expressed in human embryonic kidney 293 cells. Furthermore, we demonstrate that in the two-hybrid assay, both Shc and Sck SH2 domains can associate with the related receptor Flt1.

Chimeric VEGFRs are activated by a small-molecule dimerizer and mediate downstream signalling cascades in endothelial cells.

Despite much interest in vascular endothelial growth factor (VEGF) and its receptors (VEGFRs -1 and -2), VEGF-induced signalling cascades remain incompletely defined. Attempts to assign individual responses to a particular receptor have used either transfected cell lines, receptor-specific growth factors or antisense oligonucleotides. Such studies have attributed the majority of VEGF-induced responses to activation of VEGFR-2. As a consequence of poor growth factor-induced VEGFR-1 autophosphorylation however, observations from these studies may instead reflect the relative activation of the two receptors. We have generated novel chimeric VEGF receptors in which the dimerization domain of the B subunit of DNA gyrase is fused to the cytoplasmic domain of VEGFRs -1 and -2. When expressed in porcine aortic endothelial cells, both chimeric VEGFR-1 and -2 autophosphorylate in response to addition of the small-molecule dimerizing agent, coumermycin. Once activated, both receptors induce downstream signalling cascades, exemplified here by the activation of MAPK, PLCγ and PKB/Akt. Furthermore, we demonstrate that the Y1175 residue of VEGFR-2 is essential for the activation of PLCγ mediated by this chimeric receptor. In contrast to previous reports which show a limited ability of VEGFR-1 to mediate signalling cascades, we show that once sufficiently activated, VEGFR-1 signals in a similar manner to VEGFR-2 in endothelial cells.

Activation of ASK1, downstream MAPKK and MAPK isoforms during cardiac ischaemia

p38 MAPK is activated potently during cardiac ischaemia, although the precise mechanism by which it is activated is unclear. We used the isolated perfused rat heart to investigate the signalling pathways activated upstream of p38 during global cardiac ischaemia. Ischaemia strongly activated p38α but not the JNK pathway. The MAPKKs, MKK3, MKK4 and MKK6 have previously been identified as potential upstream activators of p38; however, in the ischaemic perfused heart, we saw activation of MKK3 and MKK6 but not MKK4. MKK3 and MKK6 showed different temporal patterns of activity, indicating distinct modes of activation and physiological function. Consistent with a lack of JNK activation, we saw no activation of MKK4 or MKK7 at any time point during ischaemia. A lack of MKK4 activation indicates, at least in the ischaemic heart, that MKK4 is not a physiologically relevant activator of p38. The MAPKKK, ASK1, was strongly activated late during ischaemia, with a similar time course to that of MKK6 and in ischaemic neonatal cardiac myocytes ASK1 expression preferentially activated MKK6 rather than MKK3. These observations suggest that during ischaemia ASK1 is coupled to p38 activation primarily via MKK6. Potent activation of ASK1 during ischaemia without JNK activation shows that during cardiac ischaemia, ASK1 preferentially activates the p38 pathway. These results demonstrate a specificity of responses seldom seen in previous studies and illustrate the benefits of using direct assays in intact tissues responding to physiologically relevant stimuli to unravel the complexities of MAPK signalling.

A nuclear export signal and oxidative stress regulate ShcD subcellular localisation: A potential role for ShcD in the nucleus

Tumour cells alter their gene expression profile to acquire a more invasive and resistant phenotype. Overexpression of the signalling adaptor protein ShcD in melanoma was found to be a prerequisite for melanoma migration and invasion. In common with other Shc proteins, ShcD has been shown to be involved in coupling receptor tyrosine kinases to the Ras-mitogen activated protein kinase signalling pathway, and to have a predominant cytoplasmic distribution. Here we report that ShcD can exist within the nucleus, and show that its CH2 domain has a critical role in nuclear export of ShcD. Analysis of GFP-tagged ShcD mutants containing deletions or amino acid substitutions within the CH2 domain revealed (83)LCTLIPRM(90) as a functional nuclear export signal. We have further demonstrated that ShcD accumulates in the nucleus upon hydrogen peroxide treatment in FLAG-ShcD expressing HEK293 cells, as well as 518.A2 melanoma cells. Cross linking experiments showed that a proportion of ShcD is associated with DNA. Moreover we have shown that ShcD fused to the GAL4 DNA binding domain can drive transcription of a GAL4 site-driven luciferase reporter, suggesting a role for ShcD in regulating gene transcription. We suggest that ShcD nuclear translocation might provide melanoma cells with a mechanism that enables them to resist DNA damage due to oxidative stress.

The role of the ShcD and RET interaction in neuroblastoma survival and migration

Preliminary screening data showed that the ShcD adaptor protein associates with the proto-oncogene RET receptor tyrosine kinase. In the present study, we aimed to investigate the molecular interaction between ShcD and RET in human neuroblastoma cells and study the functional impact of this interaction. We were able to show that ShcD immunoprecipitated with RET from SK-N-AS neuroblastoma cell lysates upon GDNF treatment. This result was validated by ShcD-RET co-localization, which was visualized using a fluorescence microscope. ShcD-RET coexpression promoted ShcD and RET endosomal localization, resulting in unexpected inhibition of the downstream ERK and AKT pathways. Interestingly, ShcD-RET association reduced the viability and migration of SK-N-AS cells. Although ShcD was previously shown to trigger melanoma cell migration and tumorigenesis, our data showed an opposite role for ShcD in neuroblastoma SK-N-AS cells via its association with RET in GDNF-treated cells. In conclusion, ShcD acts as a switch molecule that promotes contrasting biological responses depending on the stimulus ad cell type.

Insights into the Shc Family of Adaptor Proteins

The Shc family of adaptor proteins is a group of proteins that lacks intrinsic enzymatic activity. Instead, Shc proteins possess various domains that allow them to recruit different signalling molecules. Shc proteins help to transduce an extracellular signal into an intracellular signal, which is then translated into a biological response. The Shc family of adaptor proteins share the same structural topography, CH2-PTB-CH1-SH2, which is more than an isoform of Shc family proteins; this structure, which includes multiple domains, allows for the posttranslational modification of Shc proteins and increases the functional diversity of Shc proteins. The deregulation of Shc proteins has been linked to different disease conditions, including cancer and Alzheimer’s, which indicates their key roles in cellular functions. Accordingly, a question might arise as to whether Shc proteins could be targeted therapeutically to correct their disturbance. To answer this question, thorough knowledge must be acquired; herein, we aim to shed light on the Shc family of adaptor proteins to understand their intracellular role in normal and disease states, which later might be applied to connote mechanisms to reverse the disease state.

Transcriptional and Post-Translational Targeting of Myocyte Stress Protein 1 (MS1) by the JNK Pathway in Cardiac Myocytes

Myocyte Stress Protein 1 (MS1) is a muscle-specific, stress-responsive, regulator of gene expression. It was originally identified in embryonic mouse heart which showed increased expression in a rat model of left ventricular hypertrophy. To determine if MS1 was responsive to other stresses relevant to cardiac myocyte function, we tested if it could be induced by the metabolic stresses associated with ischaemia/reperfusion injury in cardiac myocytes. We found that metabolic stress increased MS1 expression, both at the mRNA and protein level, concurrent with activation of the c-Jun N-terminal Kinase (JNK) signalling pathway. MS1 induction by metabolic stress was blocked by both the transcription inhibitor actinomycin D and a JNK inhibitor, suggesting that activation of the JNK pathway during metabolic stress in cardiac myocytes leads to transcriptional induction of MS1. MS1 was also found to be an efficient JNK substrate in vitro, with a major JNK phosphorylation site identified at Thr-62. In addition, MS1 was found to co-precipitate with JNK, and inspection of the amino acid sequence upstream of the phosphorylation site, at Thr-62, revealed a putative Mitogen-Activated Protein Kinase (MAPK) binding site. Taken together, these data identify MS1 as a likely transcriptional and post-translational target for the JNK pathway in cardiac myocytes subjected to metabolic stress.