AU-rich elements (AREs) in the 3' untranslated region (UTR) of unstable mRNAs dictate their degradation. An RNAi-based screen performed in Drosophila S2 cells has revealed that Dicer1, Argonaute1 (Ago1) and Ago2, components involved in microRNA (miRNA) processing and function, are required for the rapid decay of mRNA containing AREs of tumor necrosis factor-alpha. The requirement for Dicer in the instability of ARE-containing mRNA (ARE-RNA) was confirmed in HeLa cells. We further observed that miR16, a human miRNA containing an UAAAUAUU sequence that is complementary to the ARE sequence, is required for ARE-RNA turnover. The role of miR16 in ARE-RNA decay is sequence-specific and requires the ARE binding protein tristetraprolin (TTP). TTP does not directly bind to miR16 but interacts through association with Ago/eiF2C family members to complex with miR16 and assists in the targeting of ARE. miRNA targeting of ARE, therefore, appears to be an essential step in ARE-mediated mRNA degradation.

Overexpression of the ErbB2 receptor, a major component of the ErbB receptor signaling network, contributes to the development of a number of human cancers. ErbB2 presents itself, therefore, as a target for antibody-mediated therapies. In this respect, anti-ErbB2 monoclonal antibody 4D5 specifically inhibits the growth of tumor cells overexpressing ErbB2. We have analyzed the effect of 4D5-mediated ErbB2 inhibition on the cell cycle of the breast tumor cell line BT474. 4D5 treatment of BT474 cells resulted in a G 1 arrest, preceded by rapid dephosphorylation of ErbB2, inhibition of cytoplasmic signal transduction pathways, accumulation of the cyclin-dependent kinase inhibitor p27 Kip1 , and inactivation of cyclin-Cdk2 complexes. Kip1 accumulation, p27 Kip1 redirection onto Cdk2 complexes is sufficient to inactivate Cdk2 and establish the G 1 block. These data suggest that ErbB2 overexpression leads to potentiation of cyclin E-Cdk2 activity through regulation of p27 Kip1 sequestration proteins, thus deregulating the G 1 /S transition. Moreover, through comparison with an ErbB2-overexpressing cell line insensitive to 4D5 treatment, we demonstrate the specificity of these cell cycle events and show that ErbB2 overexpression alone is insufficient to determine the cellular response to receptor inhibition.The ErbB family of type I receptor tyrosine kinases has four members, ErbB1/epidermal growth factor receptor, ErbB2/ Neu, ErbB3, and ErbB4. Although these receptors share common structural elements, including an extracellular ligandbinding domain and an intracellular tyrosine kinase domain, ligands have been identified only for ErbB1, ErbB3, and ErbB4 (for a review, see reference 16). ErbB2 remains an orphan receptor, with no diffusible ErbB2-specific ligand identified. However, ErbB2 can be transactivated through heterodimerization with other ErbB family members (11, 62) and appears to be their preferred heterodimerization partner (23, 30). ErbB2-containing heterodimers couple potently to major mitogenic signaling cascades, such as the mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase (PI3-kinase) pathways (16). Moreover, ErbB2 plays a role in the potentiation and prolongation of ErbB receptor signaling (4,22,30,49).The role of growth factors and their cognate receptors in cell growth and differentiation is now well established. Additionally, deregulation of growth factor receptors and/or elements of their signaling pathways occurs during the stepwise progression of a normal cell to a malignant phenotype. In this respect, two ErbB family members, ErbB1 and ErbB2, are involved in the development of many human cancers, including ovary and breast cancers. Indeed, amplification of the gene encoding ErbB2, leading to overexpression of the receptor, was one of the first consistent genetic alterations found in primary human breast tumors (6,70,71). Furthermore, overexpression of ErbB2 correlates with a poor patient prognosis not only in breast cancer (24,59,70,71) but also in other malignancies, suc...

The receptor for advanced glycation end products (RAGE) is thought to be involved in the pathogenesis of a broad range of inflammatory, degenerative and hyperproliferative diseases. It binds to diverse ligands and activates multiple intracellular signaling pathways. Despite these pivotal functions, molecular events just downstream of ligand-activated RAGE have been surprisingly unknown. Here we show that the cytoplasmic domain of RAGE is phosphorylated at Ser391 by PKCζ upon binding of ligands. TIRAP and MyD88, which are known to be adaptor proteins for Toll-like receptor-2 and -4 (TLR2/4), bound to the phosphorylated RAGE and transduced a signal to downstream molecules. Blocking of the function of TIRAP and MyD88 largely abrogated intracellular signaling from ligand-activated RAGE. Our findings indicate that functional interaction between RAGE and TLRs coordinately regulates inflammation, immune response and other cellular functions.

Two modes of separation coupled with MS enable researchers to study complicated biological structures.Proteins are the molecular products of genes and play a central role in many biological processes. Protein expression occurs as a function of cellular and environmental conditions, and consequently proteins are expressed at different times and under different conditions. Insight is thus garnered by determining how protein expression has changed in a biological context.Over the past two decades, MS has become an important tool for the analysis of proteins. 1,2 One current method for the analysis of protein mixtures is proteolytic digestion followed by LC/MS/ MS. Once hard-to-handle proteins are converted into chemically well-behaved peptides, the approach overcomes many difficulties associated with protein mixture analysis. 3 Tandem MS has been particularly effective because the data can be directly used to identify peptides and subsequently infer which proteins (digested or undigested) are in the mixture. 4 This type of approach for the analysis of protein mixtures is often referred to as "shotgun" proteomics ( Figure 1).Because increasingly complicated biological structures are studied by tandem MS, the need for more powerful and highly resolving separation methods has grown. Consequently, the development and use of multidimensional LC (MDLC) in proteomics has thrived over the past few years. MDLC combines two or more forms of LC to increase the peak capacity, and thus the resolving power, of separations to better fractionate peptides prior to entering the mass spectrometer.High-resolution separation serves two functions in combination with MS. It better resolves peptides differing in charge and hydrophobicity to minimize ion suppression and improve ionization efficiency, and it simplifies the complexity of peptide ions entering the mass spectrometer to minimize undersampling. This last aspect is important because the tandem MS process is driven by data-dependent data acquisition and has a finite cycle time. A higher peak capacity and better resolving power improve the acquisition of data and can lead to a better representation of the proteins in the mixture. Improved resolution also results in a larger dynamic range.Although MDLC is by no means a new concept and has a long history, it has been enjoying a renaissance in proteomics. 5 In this article, we will provide an overview of the background, important applications, and future prospects for MDLC; in particular, we will focus on applications involving peptides. (To listen to a podcast about this feature, please go to the Analytical Chemistry website at pubs.acs.org/ac.)

The calcium-binding proteins S100A8 and S100A9 can dimerize to form calprotectin, the release of which during tissue damage has been implicated in inflammation and metastasis. However, receptor(s) mediating the physiologic and pathophysiologic effects of this damage-associated "danger signal" are uncertain. In this study, searching for candidate calprotectin receptors by affinity isolation-mass spectrometry, we identified the cell surface glycoprotein EMMPRIN/BASIGIN (CD147/BSG). EMMPRIN specifically bound to S100A9 but not S100A8. Induction of cytokines and matrix metalloproteases (MMP) by S100A9 was markedly downregulated in melanoma cells by attenuation of EMMPRIN. We found that EMMPRIN signaling used the TNF receptorassociated factor TRAF2 distinct from the known S100-binding signaling pathway mediated by RAGE (AGER). S100A9 strongly promoted migration when EMMPRIN was highly expressed, independent of RAGE, whereas EMMPRIN blockade suppressed migration by S100A9. Immunohistologic analysis of melanomas revealed that EMMPRIN was expressed at both the invasive edge of lesions and the adjacent epidermis, where S100A9 was also strongly expressed. In epidermal-specific transgenic mice, tail vein-injected melanoma accumulated in skin expressing S100A9 but not S100A8. Together, our results establish EMMPRIN as a receptor for S100A9 and suggest the therapeutic use in targeting S100A9-EMMPRIN interactions. Cancer Res; 73(1); 172-83. Ó2012 AACR.