Walter Kolch

MAP kinase signalling pathways in cancer

Cancer can be perceived as a disease of communication between and within cells. The aberrations are pleiotropic, but mitogen-activated protein kinase (MAPK) pathways feature prominently. Here, we discuss recent findings and hypotheses on the role of MAPK pathways in cancer. Cancerous mutations in MAPK pathways are frequently mostly affecting Ras and B-Raf in the extracellular signal-regulated kinase pathway. Stress-activated pathways, such as Jun N-terminal kinase and p38, largely seem to counteract malignant transformation. The balance and integration between these signals may widely vary in different tumours, but are important for the outcome and the sensitivity to drug therapy.

Coordinating ERK/MAPK signalling through scaffolds and inhibitors

The pathway from Ras through Raf and MEK (MAPK and ERK kinase) to ERK/MAPK (extracellular signal-regulated kinase/mitogen-activated protein kinase) regulates many fundamental cellular processes. Recently, a number of scaffolding proteins and endogenous inhibitors have been identified, and their important roles in regulating signalling through this pathway are now emerging. Some scaffolds augment the signal flux, but also mediate crosstalk with other pathways; certain adaptors target MEK–ERK/MAPK complexes to subcellular localizations; others provide regulated inhibition. Computational modelling indicates that, together, these modulators can determine the dynamic biological behaviour of the pathway.

Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP

Raf-1 phosphorylates and activates MEK-1, a kinase that activates the extracellular signal regulated kinases (ERK). This kinase cascade controls the proliferation and differentiation of different cell types. Here we describe a Raf-1-interacting protein, isolated using a yeast two-hybrid screen. This protein inhibits the phosphorylation and activation of MEK by Raf-1 and is designated RKIP (Raf kinase inhibitor protein). In vitro, RKIP binds to Raf-1, MEK and ERK, but not to Ras. RKIP co-immunoprecipitates with Raf-1 and MEK from cell lysates and colocalizes with Raf-1 when examined by confocal microscopy. RKIP is not a substrate for Raf-1 or MEK, but competitively disrupts the interaction between these kinases. RKIP overexpression interferes with the activation of MEK and ERK, induction of AP-1-dependent reporter genes and transformation elicited by an oncogenically activated Raf-1 kinase. Downregulation of endogenous RKIP by expression of antisense RNA or antibody microinjection induces the activation of MEK-, ERK- and AP-1-dependent transcription. RKIP represents a new class of protein-kinase-inhibitor protein that regulates the activity of the Raf/MEK/ERK module.

Signalling ballet in space and time

Although we have amassed extensive catalogues of signalling network components, our understanding of the spatiotemporal control of emergent network structures has lagged behind. Dynamic behaviour is starting to be explored throughout the genome, but analysis of spatial behaviours is still confined to individual proteins. The challenge is to reveal how cells integrate temporal and spatial information to determine specific biological functions. Key findings are the discovery of molecular signalling machines such as Ras nanoclusters, spatial activity gradients and flexible network circuitries that involve transcriptional feedback. They reveal design principles of spatiotemporal organization that are crucial for network function and cell fate decisions.

Identification of the Mechanisms Regulating the Differential Activation of the MAPK Cascade by Epidermal Growth Factor and Nerve Growth Factor in PC12 Cells

In PC12 cells, epidermal growth factor (EGF) transiently stimulates the mitogen-activated protein (MAP) kinases, ERK1 and ERK2, and provokes cellular proliferation. In contrast, nerve growth factor (NGF) stimulation leads to the sustained activation of the MAPKs and subsequently to neuronal differentiation. It has been shown that both the magnitude and longevity of MAPK activation governs the nature of the cellular response. The activations of MAPKs are dependent upon two distinct small G-proteins, Ras and Rap1, that link the growth factor receptors to the MAPK cascade by activating c-Raf and B-Raf, respectively. We found that Ras was transiently stimulated upon both EGF and NGF treatment of PC12 cells. However, EGF transiently activated Rap1, whereas NGF stimulated prolonged Rap1 activation. The activation of the ERKs was due almost exclusively (>90%) to the action of B-Raf. The transient activation of the MAPKs by EGF was a consequence of the formation of a short lived complex assembling on the EGF receptor itself, composed of Crk, C3G, Rap1, and B-Raf. In contrast, NGF stimulation of the cells resulted in the phosphorylation of FRS2. FRS2 scaffolded the assembly of a stable complex of Crk, C3G, Rap1, and B-Raf resulting in the prolonged activation of the MAPKs. Together, these data provide a signaling link between growth factor receptors and MAPK activation and a mechanistic explanation of the differential MAPK kinetics exhibited by these growth factors.

Mechanism of Suppression of the Raf/MEK/Extracellular Signal-Regulated Kinase Pathway by the Raf Kinase Inhibitor Protein

ABSTRACT We have recently identified the Raf kinase inhibitor protein (RKIP) as a physiological endogenous inhibitor of the Raf-1/MEK/extracellular signal-regulated kinase (ERK) pathway. RKIP interfered with MEK phosphorylation and activation by Raf-1, resulting in the suppression of both Raf-1-induced transformation and AP-1-dependent transcription. Here we report the molecular mechanism of RKIPs inhibitory function. RKIP can form ternary complexes with Raf-1, MEK, and ERK. However, whereas MEK and ERK can simultaneously associate with RKIP, Raf-1 binding to RKIP and that of MEK are mutually exclusive. RKIP is able to dissociate a Raf-1–MEK complex and behaves as a competitive inhibitor of MEK phosphorylation. Mapping of the binding domains showed that MEK and Raf-1 bind to overlapping sites in RKIP, whereas MEK and RKIP associate with different domains in Raf-1, and Raf-1 and RKIP bind to different sites in MEK. Both the Raf-1 and the MEK binding sites in RKIP need to be destroyed in order to relieve RKIP-mediated suppression of the Raf-1/MEK/ERK pathway, indicating that binding of either Raf-1 or MEK is sufficient for inhibition. The properties of RKIP reveal the specific sequestration of interacting components as a novel motif in the cells repertoire for the regulation of signaling pathways.

Epstein-Barr virus latent membrane protein-1 triggers AP-1 activity via the c-Jun N-terminal kinase cascade.

The Epstein–Barr virus latent membrane protein‐1 (LMP‐1) is an integral membrane protein which transforms fibroblasts and is essential for EBV‐mediated B‐cell immortalization. LMP‐1 has been shown to trigger cellular NF‐κB activity which, however, cannot fully explain the oncogenic potential of LMP‐1. Here we show that LMP‐1 induces the activity of the AP‐1 transcription factor, a dimer of Jun/Jun or Jun/Fos proteins. LMP‐1 effects on AP‐1 are mediated through activation of the c‐Jun N‐terminal kinase (JNK) cascade, but not the extracellular signal‐regulated kinase (Erk) pathway. Consequently, LMP‐1 triggers the activity of the c‐Jun N‐terminal transactivation domain which is known to be activated upon JNK‐mediated phosphorylation. Deletion analysis indicates that the 55 C‐terminal amino acids of the LMP‐1 molecule, but not its TRAF interaction domain, are essential for AP‐1 activation. JNK‐mediated transcriptional activation of AP‐1 is the direct output of LMP‐1‐triggered signaling, as shown by an inducible LMP‐1 mutant. Using a tetracycline‐regulated LMP‐1 allele, we demonstrate that JNK is also an effector of non‐cytotoxic LMP‐1 signaling in B cells, the physiological target cells of EBV. In summary, our data reveal a novel effector of LMP‐1, the SEK/JNK/c‐Jun/AP‐1 pathway, which contributes to our understanding of the immortalizing and transforming potential of LMP‐1.

MECHANISM OF INHIBITION OF RAF-1 BY PROTEIN KINASE A

The cytoplasmic Raf-1 kinase is essential for mitogenic signalling by growth factors, which couple to tyrosine kinases, and by tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate, which activate protein kinase C (PKC). Signalling by the Raf-1 kinase can be blocked by activation of the cyclic AMP (cAMP)-dependent protein kinase A (PKA). The molecular mechanism of this inhibition is not precisely known but has been suggested to involve attenuation of Raf-1 binding to Ras. Using purified proteins, we show that in addition to weakening the interaction of Raf-1 with Ras, PKA can inhibit Raf-1 function directly via phosphorylation of the Raf-1 kinase domain. Phosphorylation by PKA interferes with the activation of Raf-1 by either PKC alpha or the tyrosine kinase Lck and even can downregulate the kinase activity of Raf-1 previously activated by PKC alpha or amino-terminal truncation. This type of inhibition can be dissociated from the ability of Raf-1 to associate with Ras, since (i) the isolated Raf-1 kinase domain, which lacks the Ras binding domain, is still susceptible to inhibition by PKA, (ii) phosphorylation of Raf-1 by PKC alpha alleviates the PKA-induced reduction of Ras binding but does not prevent the downregulation of Raf-1 kinase activity by PKA and (iii) cAMP agonists antagonize transformation by v-Raf, which is Ras independent.

Raf Kinase Inhibitor Protein Interacts with NF-κB-Inducing Kinase and TAK1 and Inhibits NF-κB Activation

ABSTRACT The Raf kinase inhibitor protein (RKIP) acts as a negative regulator of the mitogen-activated protein (MAP) kinase (MAPK) cascade initiated by Raf-1. RKIP inhibits the phosphorylation of MAP/extracellular signal-regulated kinase 1 (MEK1) by Raf-1 by disrupting the interaction between these two kinases. We show here that RKIP also antagonizes the signal transduction pathways that mediate the activation of the transcription factor nuclear factor kappa B (NF-κB) in response to stimulation with tumor necrosis factor alpha (TNF-α) or interleukin 1 beta. Modulation of RKIP expression levels affected NF-κB signaling independent of the MAPK pathway. Genetic epistasis analysis involving the ectopic expression of kinases acting in the NF-κB pathway indicated that RKIP acts upstream of the kinase complex that mediates the phosphorylation and inactivation of the inhibitor of NF-κB (IκB). In vitro kinase assays showed that RKIP antagonizes the activation of the IκB kinase (IKK) activity elicited by TNF-α. RKIP physically interacted with four kinases of the NF-κB activation pathway, NF-κB-inducing kinase, transforming growth factor beta-activated kinase 1, IKKα, and IKKβ. This mode of action bears striking similarities to the interactions of RKIP with Raf-1 and MEK1 in the MAPK pathway. Emerging data from diverse organisms suggest that RKIP and RKIP-related proteins represent a new and evolutionarily highly conserved family of protein kinase regulators. Since the MAPK and NF-κB pathways have physiologically distinct roles, the function of RKIP may be, in part, to coordinate the regulation of these pathways.

Clinical proteomics: A need to define the field and to begin to set adequate standards

The aim of this manuscript is to initiate a constructive discussion about the definition of clinical proteomics, study requirements, pitfalls and (potential) use. Furthermore, we hope to stimulate proposals for the optimal use of future opportunities and seek unification of the approaches in clinical proteomic studies. We have outlined our collective views about the basic principles that should be considered in clinical proteomic studies, including sample selection, choice of technology and appropriate quality control, and the need for collaborative interdisciplinary efforts involving clinicians and scientists. Furthermore, we propose guidelines for the critical aspects that should be included in published reports. Our hope is that, as a result of stimulating discussion, a consensus will be reached amongst the scientific community leading to guidelines for the studies, similar to those already published for mass spectrometric sequencing data. We contend that clinical proteomics is not just a collection of studies dealing with analysis of clinical samples. Rather, the essence of clinical proteomics should be to address clinically relevant questions and to improve the state‐of‐the‐art, both in diagnosis and in therapy of diseases.