Alyson K. Freeman

14-3-3 Proteins: Diverse functions in cell proliferation and cancer progression

The 14-3-3 proteins were the first phosphoserine/phosphothreonine-binding proteins to be discovered, a finding that provided the foundation for their prominent role in cell signaling. 14-3-3 family members interact with a wide spectrum of proteins including transcription factors, biosynthetic enzymes, cytoskeletal proteins, signaling molecules, apoptosis factors, and tumor suppressors. The interaction with 14-3-3 can have a profound effect on a target protein, altering its localization, stability, conformation, phosphorylation state, activity, and/or molecular interactions. Thus, by modulating the function of a diverse array of binding partners, 14-3-3 proteins have become key regulatory components in many vital cellular processes - processes that are crucial for normal growth and development and that often become dysregulated in human cancer. This review will examine the recent advances that further elucidate the role of 14-3-3 proteins in normal growth and cancer signaling with a particular emphasis on the signaling pathways that impact cell proliferation, cell migration, and epithelial-to-mesenchymal transition.

Effects of Raf Dimerization and its Inhibition on Normal and Disease-associated Raf Signaling

Raf kinases are essential for normal Ras-Raf-MEK-ERK pathway signaling, and activating mutations in components of this pathway are associated with a variety of human cancers, as well as the related developmental disorders Noonan, LEOPARD, and cardiofaciocutaneous syndromes. Although the Raf kinases are known to dimerize during normal and disease-associated Raf signaling, the functional significance of Raf dimerization has not been fully elucidated. Here, using mutational analysis and a peptide inhibitor, we show that dimerization is required for normal Ras-dependent Raf activation and for the biological function of disease-associated Raf mutants with moderate, low, or impaired kinase activity. However, dimerization is not needed for the function of B-Raf mutants with high catalytic activity, such as V600E-B-Raf. Importantly, we find that a dimer interface peptide can effectively block Raf dimerization and inhibit Raf signaling when dimerization is required for Raf function, thus identifying the Raf dimer interface as a therapeutic target.

Phosphatases in the cellular response to DNA damage

In the last fifteen years, rapid progress has been made in delineating the cellular response to DNA damage. The DNA damage response network is composed of a large number of proteins with different functions that detect and signal the presence of DNA damage in order to coordinate DNA repair with a variety of cellular processes, notably cell cycle progression. This signal, which radiates from the chromatin template, is driven primarily by phosphorylation events, mainly on serine and threonine residues. While we have accumulated detailed information about kinases and their substrates our understanding of the role of phosphatases in the DNA damage response is still preliminary. Identifying the phosphatases and their regulation will be instrumental to obtain a complete picture of the dynamics of the DNA damage response. Here we give an overview of the DNA damage response in mammalian cells and then review the data on the role of different phosphatases and discuss their biological relevance.

The importance of Raf dimerization in cell signaling.

The Raf family of protein kinases are key signaling intermediates, acting as a central link between the membrane-bound Ras GTPases and the downstream kinases MEK and ERK. Raf kinase regulation is well-known for its complexity but only recently has it been realized that many of the mechanisms involved in Raf regulation also modulate Raf dimerization, now acknowledged to be a required step for Raf signaling in multiple cellular contexts. Recent studies have shown that Raf dimerization is necessary for normal Ras-dependent Raf kinase activation and contributes to the pathogenic function of disease-associated mutant Raf proteins with all but high intrinsic kinase activity. Raf dimerization has also been found to alter therapeutic responses and disease progression in patients treated with ATP-competitive Raf inhibitors as well as certain other kinase-targeted drugs. This demonstration of clinical significance has stimulated the recent development of biosensor assays that can monitor inhibitor-induced Raf dimerization as well as studies demonstrating the therapeutic potential of blocking Raf dimerization.

Complexity in KSR function revealed by Raf inhibitor and KSR structure studies.

The Ras, Raf, MEK and ERK proteins form an essential signal transduction pathway that is aberrantly activated in many human cancers. Kinase Suppressor of Ras (KSR) is a conserved positive modulator of this pathway, and since its discovery, there has been a concerted effort to elucidate KSR function in both normal and aberrant Ras/ERK signaling. The KSR proteins possess a C-terminal region that is closely related to the Raf family kinase domain; however, mammalian KSR proteins lack a key catalytic residue, suggesting a role as a pseudokinase. Like many other pseudokinases, KSR has scaffolding activities and interacts with Raf, MEK, and ERK to provide spatio-temporal regulation of ERK activation. Recently, significant advances have been made that further our understanding of how KSR proteins function in normal and oncogenic signaling. The newly solved KSR2/MEK1 structure has revealed important mechanistic details for how KSR regulates MEK activation and has raised questions regarding KSR kinase activity. In addition, KSR expression levels have been found to alter the effects of Raf inhibitors on oncogenic Ras/ERK signaling. Specifically, KSR1 competes with C-Raf for inhibitor-induced binding to B-Raf and in doing so attenuates the paradoxical activating effect of these drugs on ERK signaling.

Mechanisms and Potential Therapies for Acquired Resistance to Inhibitors Targeting the Raf or MEK Kinases in Cancer

Aberrant activation of the Ras/Raf/MEK/ERK signaling pathway often occurs in human cancer through the acquisition of oncogenic mutations in key pathway components. In particular, Ras mutations are found in about 20 % of human cancers, and B-Raf mutations occur in more than half of all melanomas. Thus, this pathway has become an attractive target for cancer therapies. Inhibitors targeting either the Raf or MEK kinases have shown initial success in treating cancers that depend on the Ras/Raf/MEK/ERK signaling pathway; unfortunately, resistance to the inhibitors eventually develops. For both sets of inhibitors, resistance most commonly occurs via reactivation of the Ras/Raf/MEK/ERK pathway or upregulated signaling from an alternate pathway, such as the PI3K/AKT pathway. Here, we discuss the mechanisms for acquired resistance to inhibitors targeting the Raf or MEK kinases and possible combination therapies to overcome or delay drug resistance.

Abstract 1239: Differential effects of dimerization on B-Raf and C-Raf function revealed by mutational analysis and peptide inhibitors that target the Raf dimer interface

Normal cellular function is dependent upon proper regulation of the Raf kinases, mutation of which can result in human cancer and certain developmental disorders. Inhibitors to the high activity, oncogenic V600E-B-Raf are currently in clinical use; however, caution must be taken, given that the use of these inhibitors in cells lacking V600E-B-Raf can promote heterodimerization of B-Raf and C-Raf, resulting in paradoxical pathway activation instead of inhibition. Thus, a full understanding of the Raf activation process is critical for the development of effective therapeutic strategies. Toward this end, we investigated the importance of dimerization in Raf activation and identified novel differences between B-Raf and C-Raf. In the context of normal cellular signaling, we find that growth factor stimulation induces strong B-Raf/C-Raf heterodimerization as well as some Raf homodimerization, and increases the kinase activity of both B-Raf and C-Raf. In contrast, growth factor stimulation has little effect on A-Raf. To further explore the B-Raf and C-Raf interactions, we utilized protein mutations in the dimer interface that either enhance (E586K-B-Raf and E478K-C-Raf) or prevent (R509H-B-Raf and R401H-C-Raf) Raf dimerization. Interestingly, these mutations had only a modest effect on the kinase activity of B-Raf; however, the E-K mutation greatly enhanced C-Raf activity and the R-H mutation completely abolished C-Raf kinase activity. In addition, knockdown of C-Raf was found to have little effect on growth factor-mediated B-Raf activation, whereas knockdown of B-Raf dramatically inhibited C-Raf activation following stimulation, highlighting the dependence of C-Raf activation on B-Raf. Next, we examined the effects of dimerization on mutationally-activated B-Raf and C-Raf proteins that are associated with human disease. Strikingly, we found that although alterations in the dimer interface had an impact on the ability of all the mutant proteins to heterodimerize, these alterations only affected the biological activity (as measured in focus forming assays) of B-Raf and C-Raf proteins with moderate to low kinase activity, but not B-Raf proteins possessing high kinase activity. Based on the amino acid sequence of the dimer interface region, a Raf-Dimer-Interface (RDI) peptide was designed. The RDI peptide was found to bind both B-Raf and C-Raf and could disrupt Raf heterodimerization in response to growth factor stimulation and inhibit the biological activity of mutant Raf proteins with moderate to low kinase activity. Together these data indicate that dimerization is important for Raf function under normal signaling conditions and in certain mutational settings. Moreover, targeting the Raf dimer interface represents a new inhibitor strategy with therapeutic potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1239. doi:1538-7445.AM2012-1239