No abstract

The N terminus of the human MUC2 mucin (amino acids 1-1397) has been expressed as a recombinant tagged protein in Chinese hamster ovary cells. The intracellular form was found to be an endoglycosidase H-sensitive monomer, whereas the secreted form was an oligomer that gave monomers upon disulfide bond reduction. The secreted MUC2 N terminus contained a trypsin-resistant core fragment. Edman sequencing and mass spectrometry of the peptides obtained localized this core fragment to the C-terminal end of the recombinant protein. This core retained its oligomeric nature with an apparent mass of ϳ240 kDa. Upon reduction, peptides of ϳ85 kDa were found, suggesting that the N terminus forms trimers. This interpretation was also supported by gel electrophoresis and gel filtration of the intact MUC2 N terminus. Electron microscopy revealed three globular domains each linked via an extended and flexible region to a central part in a trefoil-like manner. Immunostaining with gold-labeled antibodies localized the N-terminal end to the three globular structures, and the antibodies directed against the Myc and green fluorescent protein tags attached at the C terminus localized these to the stalk side of the central trefoil. The N terminus of the MUC2 mucin is thus assembled into trimers that contain proteolytically stable parts, suggesting that MUC2 can only be partly degraded by intestinal proteases and thus is able to maintain a mucin network protecting the intestine.Mucins are highly glycosylated proteins protecting the mucosal surfaces of the body. All mucins are characterized by their heavy O-glycosylation, which is clustered in mucin domains rich in the amino acids Ser, Thr, and Pro. Mucins can be structured in different ways, either as membrane-bound with a transmembrane domain or secreted as monomers or as polymers (1, 2). The latter mucins are also described as gel-forming, as these give the mucus its viscoelastic properties. Four human gel-forming mucins have been found, MUC2, MUC5AC, MUC5B, and MUC6, which are clustered on chromosome 11p15 (3). However, the best studied mucin is the porcine submaxillary mucin (PSM), 1 which has become a model also for the human mucins of this type (3-6).The MUC2 mucin is the main gel-forming mucin of the small and large intestines and is produced by the intestinal goblet cells (2, 7-10). It is a major structural component of the mucous barrier covering the epithelium, protecting the epithelial cells from microorganisms as well as digestive enzymes. In fact, one of the remarkable features of this mucin is that it can withstand the pancreatic digestive enzymes. That the highly glycosylated mucin domains are totally resistant to these enzymes is easy to understand. However, the globular, less glycosylated ends must also be relatively proteolytically resistant, as the polymers must remain intact to maintain the mucous gel. The MUC2 mucin is difficult to solubilize, and it is insoluble in 4 M guanidinium chloride (8, 10), a property that might be related to the appearance of nonreducible ...

Targeted drugs are less toxic than traditional chemotherapeutic therapies; however, the proportion of patients that benefit from these drugs is often smaller. A marker that confidently predicts patient response to a specific therapy would allow an individual therapy selection most likely to benefit the patient. Here, we used quantitative mass spectrometry to globally profile the basal phosphoproteome of a panel of non-small cell lung cancer cell lines. The effect of the kinase inhibitor dasatinib on cellular growth was tested against the same panel. From the phosphoproteome profiles, we identified 58 phosphorylation sites, which consistently differ between sensitive and resistant cell lines. Many of the corresponding proteins are involved in cell adhesion and cytoskeleton organization. We showed that a signature of only 12 phosphorylation sites is sufficient to accurately predict dasatinib sensitivity. The introduction of targeted drugs for treating cancer is a major biomedical achievement of the past decade (1, 2). Because these drugs selectively block molecular pathways that are typically overactivated in tumor cells, they are more precise and less toxic than traditional chemotherapeutics. However, although many cancer patients benefit from a specific targeted therapy, many others do not. Therefore, predictive molecular markers are needed to confidently predict patient response to a specific therapy. Such markers would facilitate therapy personalization, where the selected therapy is based on the molecular profile of the patient.Predictive tests currently used in the clinic are frequently based on one particular marker that is often linked to the drug target. A well known example for a predictive test is assessing HER2/neu overexpression using immunohistochemistry or fluorescent in situ hybridization to predict the response to therapy with trastuzumab (Herceptinா; Roche) (3, 4). However, in some cases the expression or mutational status of the target or other singleton markers might not be sufficient to predict a therapeutic response. Recently, several studies tried to identify molecular signatures comprising multiple markers for response predictions, usually based on gene expression profiling (5, 6). To our knowledge, no study successfully identified a signature from global phosphoproteomic profiles so far.Recent advances in mass spectrometry, methods for enriching phosphorylated proteins or peptides, and computer algorithms for analyzing proteomics data have enabled the application of mass spectrometry-based proteomics to monitor phosphorylation events in a global and unbiased manner. These methods have become sufficiently sensitive and robust to localize and quantify the phosphorylation sites within a peptide sequence (7-9). Phosphorylation events are important in signal transduction, where signals caused by external stimuli are transmitted from the cell membrane to the nucleus. Aberrations in these signal transduction pathways are particularly important for understanding the mechanisms of certain diseases,...

Bisindolylmaleimide compounds such as GF109203X are potent inhibitors of protein kinase C (PKC) activity. Although bisindolylmaleimides are not entirely selective for PKC and are known to inhibit a few other protein kinases, these reagents have been extensively used to study the functional roles of PKC family enzymes in cellular signal transduction for more than a decade. Here, we establish a proteomics approach to gain further insights into the cellular effects of this compound class. Functional immobilization of suitable bisindolylmaleimide analogues in combination with the specific purification of cellular binding proteins by affinity chromatography led to the identification of several known and previously unknown enzyme targets. Subsequent in vitro binding and activity assays confirmed the protein kinases Ste20-related kinase and cyclin-dependent kinase 2 (CDK2) and the non-protein kinases adenosine kinase and quinone reductase type 2 as novel targets of bisindolylmaleimide inhibitors. As observed specifically for CDK2, minor chemical variation of the ligand by immobilizing the closely related bisindolylmaleimides III, VIII, and X dramatically affected target binding. These observed changes in affinity correlated with both the measured IC 50 values for in vitro CDK2 inhibition and results from molecular docking into the CDK2 crystal structure. Moreover, the conditions for affinity purification could be adapted in a way that immobilized bisindolylmaleimide III selectively interacted with either PKC␣ or ribosomal S6 protein kinase 1 only after activation of these kinases. Thus, we have established an efficient technique for the rapid identification of cellular bisindolylmaleimide targets and further demonstrate the comparative selectivity profiling of closely related kinase inhibitors within a cellular proteome. Molecular & Cellular Proteomics 3:490 -500, 2004.