First identified in the early 1980s as retroviral oncogenes, the Raf proteins have been the objects of intense research. The discoveries 10 years later that the Raf family members (Raf-1, B-Raf, and A-Raf) are bona fide Ras effectors and upstream activators of the ubiquitous ERK pathway increased the interest in these proteins primarily because of the central role that this cascade plays in cancer development. The important role of Raf in cancer was corroborated in 2002 with the discovery of B-Raf genetic mutations in a large number of tumors. This led to intensified drug development efforts to target Raf signaling in cancer. This work yielded not only recent clinical successes but also surprising insights into the regulation of Raf proteins by homodimerization and heterodimerization. Surprising insights also came from the hunt for new Raf targets. Although MEK remains the only widely accepted Raf substrate, new kinase-independent roles for Raf proteins have emerged. These include the regulation of apoptosis by suppressing the activity of the proapoptotic kinases, ASK1 and MST2, and the regulation of cell motility and differentiation by controlling the activity of Rok-α. In this review, we discuss the regulation of Raf proteins and their role in cancer, with special focus on the interacting proteins that modulate Raf signaling. We also describe the new pathways controlled by Raf proteins and summarize the successes and failures in the development of efficient anticancer therapies targeting Raf. Finally, we also argue for the necessity of more systemic approaches to obtain a better understanding of how the Ras-Raf signaling network generates biological specificity.

Mitogen-activated protein kinase (MAPK) pathways represent ubiquitous signal transduction pathways that regulate all aspects of life and are frequently altered in disease. Here, we focus on the role of MAPK pathways in modulating drug sensitivity and resistance in cancer. We briefly discuss new findings in the extracellular signaling-regulated kinase (ERK) pathway, but mainly focus on the mechanisms how stress activated MAPK pathways, such as p38 MAPK and the Jun N-terminal kinases (JNK), impact the response of cancer cells to chemotherapies and targeted therapies. In this context, we also discuss the role of metabolic and epigenetic aberrations and new therapeutic opportunities arising from these changes.

Protein phosphorylation participates in the regulation of all fundamental biological processes, and protein kinases have been intensively studied. However, while the focus was on catalytic activities, accumulating evidence suggests that non-catalytic properties of protein kinases are essential, and in some cases even sufficient for their functions. These non-catalytic functions include the scaffolding of protein complexes, the competition for protein interactions, allosteric effects on other enzymes, subcellular targeting, and DNA binding. This rich repertoire often is used to coordinate phosphorylation events and enhance the specificity of substrate phosphorylation, but also can adopt functions that do not rely on kinase activity. Here, we discuss such kinase independent functions of protein and lipid kinases focussing on kinases that play a role in the regulation of cell proliferation, differentiation, apoptosis, and motility.

Western blot data are widely used in quantitative applications such as statistical testing and mathematical modelling. To ensure accurate quantitation and comparability between experiments, Western blot replicates must be normalised, but it is unclear how the available methods affect statistical properties of the data. Here we evaluate three commonly used normalisation strategies: (i) by fixed normalisation point or control; (ii) by sum of all data points in a replicate; and (iii) by optimal alignment of the replicates. We consider how these different strategies affect the coefficient of variation (CV) and the results of hypothesis testing with the normalised data. Normalisation by fixed point tends to increase the mean CV of normalised data in a manner that naturally depends on the choice of the normalisation point. Thus, in the context of hypothesis testing, normalisation by fixed point reduces false positives and increases false negatives. Analysis of published experimental data shows that choosing normalisation points with low quantified intensities results in a high normalised data CV and should thus be avoided. Normalisation by sum or by optimal alignment redistributes the raw data uncertainty in a mean-dependent manner, reducing the CV of high intensity points and increasing the CV of low intensity points. This causes the effect of normalisations by sum or optimal alignment on hypothesis testing to depend on the mean of the data tested; for high intensity points, false positives are increased and false negatives are decreased, while for low intensity points, false positives are decreased and false negatives are increased. These results will aid users of Western blotting to choose a suitable normalisation strategy and also understand the implications of this normalisation for subsequent hypothesis testing.

Introduction 3391 2. Medical Applications of Nanomaterials 3391 2.1. Use of Nanomaterials in Diagnostic Applications 3392 3. Interaction of Nanomaterials with Living Cells 3392 3.1. Role of Physicochemical NP Properties in the Interaction between NPs and Biological Systems 3393 3.2. NP Uptake into the Cells 3393 3.2.1. Role of Size and Shape in NP Uptake 3393 3.2.2. Role of Surface Charge and Protein Corona on NP Uptake 3395 3.3. Activation of Signal Processing Pathways by NPs 3397 3.3.1. Interaction of NPs with Cell Membrane Receptors 3397 3.3.2. Role of Oxidative Stress and ROS in NP-Induced Signaling 3398 3.3.3. NPs Induce Cell Death Pathways 3400 3.3.4. Activation of Mechanotransduction Receptors by Magnetic NPs 3400 4. Magnetic NPs and Cell Signaling 3400 4.1. Controlled Activation of Cell Surface Receptors by Magnetic NPs 3400 4.1.1. Activation of Signal Transduction Receptors by Magnetic NPs 3400 4.1.2. Activation of Mechanotransduction Receptors and Mechanosensitive Signaling Pathways by Magnetic NPs 3400 4.2. Pathway Activation by Magnetic-NP-Induced Hyperthermia 3401 4.2.1. Molecular Targets of Hyperthermia: Heat Shock Proteins and p53 3401 5. Conclusions 3402 Author Information 3402 Corresponding Author 3402 Present Address 3402 Notes 3402 Biographies 3402 Acknowledgments 3403 References 3403

BackgroundCell-to-cell variability in protein expression can be large, and its propagation through signaling networks affects biological outcomes. Here, we apply deterministic and probabilistic models and biochemical measurements to study how network topologies and cell-to-cell protein abundance variations interact to shape signaling responses.ResultsWe observe bimodal distributions of extracellular signal-regulated kinase (ERK) responses to epidermal growth factor (EGF) stimulation, which are generally thought to indicate bistable or ultrasensitive signaling behavior in single cells. Surprisingly, we find that a simple MAPK/ERK-cascade model with negative feedback that displays graded, analog ERK responses at a single cell level can explain the experimentally observed bimodality at the cell population level. Model analysis suggests that a conversion of graded input–output responses in single cells to digital responses at the population level is caused by a broad distribution of ERK pathway activation thresholds brought about by cell-to-cell variability in protein expression.ConclusionsOur results show that bimodal signaling response distributions do not necessarily imply digital (ultrasensitive or bistable) single cell signaling, and the interplay between protein expression noise and network topologies can bring about digital population responses from analog single cell dose responses. Thus, cells can retain the benefits of robustness arising from negative feedback, while simultaneously generating population-level on/off responses that are thought to be critical for regulating cell fate decisions.

A-Raf belongs to the family of oncogenic Raf kinases that are involved in mitogenic signaling by activating the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK pathway. Low kinase activity of A-Raf toward MEK suggested that A-Raf might have alternative functions. Here, we show that A-Raf prevents cancer cell apoptosis contingent on the expression of the heterogeneous nuclear ribonucleoprotein H (hnRNP H) splice factor, which is required for the correct transcription and expression of a-raf. Apoptosis was prevented by A-Raf through sequestration and inactivation of the proapoptotic MST2 kinase. Small interfering RNA-mediated knockdown of hnRNP H or A-Raf resulted in MST2-dependent apoptosis. In contrast, enforced expression of either hnRNP H or A-Raf partially counteracted apoptosis induced by etoposide. In vivo expression studies of colon specimens corroborated the overexpression of hnRNP H in malignant tissues and its correlation with A-Raf levels. Our findings define a novel mechanism that is usurped in tumor cells to escape naturally imposed apoptotic signals. Cancer Res; 70(4); 1679-88. ©2010 AACR.

Clinically used RAF inhibitors are ineffective in RAS mutant tumors because they enhance homo- and heterodimerization of RAF kinases, leading to paradoxical activation of ERK signaling. Overcoming enhanced RAF dimerization and the resulting resistance is a challenge for drug design. Combining multiple inhibitors could be more effective, but it is unclear how the best combinations can be chosen. We built a next-generation mechanistic dynamic model to analyze combinations of structurally different RAF inhibitors, which can efficiently suppress MEK/ERK signaling. This rule-based model of the RAS/ERK pathway integrates thermodynamics and kinetics of drug-protein interactions, structural elements, posttranslational modifications, and cell mutational status as model rules to predict RAF inhibitor combinations for inhibiting ERK activity in oncogenic RAS and/or BRAFV600E backgrounds. Predicted synergistic inhibition of ERK signaling was corroborated by experiments in mutant NRAS, HRAS, and BRAFV600E cells, and inhibition of oncogenic RAS signaling was associated with reduced cell proliferation and colony formation.