Using DNA microarrays together with quantitative proteomic techniques (ICAT reagents, two-dimensional DIGE, and MS), we evaluated the correlation of mRNA and protein levels in two hematopoietic cell lines representing distinct stages of myeloid differentiation, as well as in the livers of mice treated for different periods of time with three different peroxisome proliferative activated receptor agonists. We observe that the differential expression of mRNA (up or down) can capture at most 40% of the variation of protein expression. Although the overall pattern of protein expression is similar to that of mRNA expression, the incongruent expression between mRNAs and proteins emphasize the importance of posttranscriptional regulatory mechanisms in cellular development or perturbation that can be unveiled only through integrated analyses of both proteins and mRNAs. Molecular & Cellular Proteomics 3:960 -969, 2004.Genome-wide mRNA expression profiling by means of DNA microarrays has proven to be a powerful approach in characterizing the changes in biological processes such as disease states, developmental stages, and responses to drugs or genetic perturbations (1). However, DNA arrays measure only the changes at the mRNA level. Most biological functions are executed by the proteins rather than mRNAs. While the expression of many genes is controlled at the transcriptional level, other genes also employ posttranscriptional regulation processes involving mRNA stability, translation initiation, and protein stability. An important issue is the extent to which the changing expression patterns of mRNAs reflect corresponding changes in their cognate proteins. Recent advances in quantitative proteomics, especially the application of ICAT reagents in conjunction with MS, have made possible simultaneous quantitative comparison of hundreds of proteins between two complex mixtures (2). Integrated analyses of mRNA and protein expression data by concurrent measurement of both have revealed moderate to poor correlation in yeast and Halobacteria (3-5). Discordant expression of protein and mRNA was also observed in lung adenocarcinomas (6). However, these analyses examined only one aspect of a biological system, i.e. the steady-state levels of mRNAs and proteins. Another important aspect that concerns the kinetic process of perturbation and how the correlation of mRNA and protein evolves during this process was not addressed. Here, we evaluated the correlation of mRNA and protein expression in mammalian systems under two experimental conditions. In the first, we compared steady-state levels of mRNAs and proteins between two related cell lines representing distinct hematopoietic stages, i.e. multipotent myeloid precursors versus lineage-committed promyelocytic cells. In the second condition, we used a mouse model to demonstrate the kinetic changes in liver mRNA and protein levels in response to treatment with three different drugs. In both cases, we observed a moderate correlation between mRNA and protein levels with the expression of m...

Novel potent and selective diarylimidazole inhibitors of p38 MAP (mitogen-activated protein) kinase are described which have activity in both cell-based assays of tumor necrosis factor-alpha (TNF-alpha) release and an animal model of rheumatoid arthritis. The SAR leading to the development of selectivity against c-Raf and JNK2alpha1 kinases is presented, with key features being substitution of the 4-aryl ring with m-trifluoromethyl and substitution of the 5-heteroaryl ring with a 2-amino substituent. Cell-based activity was significantly enhanced by incorporation of a 4-piperidinyl moiety at the 2-position of the imidazole which also enhanced aqueous solubility. In general, oral bioavailability of this class of compounds was found to be poor unless the imidazole was methylated on nitrogen. This work led to identification of 48, a potent (p38 MAP kinase inhibition IC50 0.24 nM) and selective p38 MAP kinase inhibitor which inhibits lipopolysaccharide-stimulated release of TNF-alpha from human blood with an IC50 2.2 nM, shows good oral bioavailability in rat and rhesus monkey, and demonstrates significant improvement in measures of disease progression in a rat adjuvant-induced arthritis model.

The adhesion molecules known as selectins mediate the capture of neutrophils from the bloodstream. We have previously reported that ligation and cross-linking of L-selectin on the neutrophil surface enhances the adhesive function of ␤ 2 -integrins in a synergistic manner with chemotactic agonists. In this work, we examined degranulation and adhesion of neutrophils in response to cross-linking of L-selectin and addition of interleukin-8. Cross-linking of L-selectin induced priming of degranulation that was similar to that observed with the alkaloid cytochalasin B. Activation mediated by L-selectin of neutrophil shape change and adhesion through CD11b/CD18 were strongly blocked by Merck C, an imidazole-based inhibitor of p38 mitogen-activated protein kinase (MAPK), but not by a structurally similar non-binding regioisomer. Priming by L-selectin of the release of secondary, tertiary, and secretory, but not primary, granules was blocked by inhibition of p38 MAPK. Peak phosphorylation of p38 MAPK was observed within 1 min of cross-linking L-selectin, whereas phosphorylation of ERK1/2 was highest at 10 min. Phosphorylation of p38 MAPK, but not ERK1/2, was inhibited by Merck C. These data suggest that signal transduction as a result of clustering L-selectin utilizes p38 MAPK to effect neutrophil shape change, integrin activation, and the release of secondary, tertiary, and secretory granules.Neutrophils circulate in the vasculature in a passive state and become more adhesive upon stimulation at sites of inflammation. Margination to the vessel wall and subsequent transmigration and phagocytosis (1) requires a number of surface proteins, including the ␤ 2 -integrins and the selectins, as mediators of adherence to the endothelium (2-5). A sequence of molecular and biophysical events has been identified that facilitates neutrophil activation and increased adherence during the acute inflammatory response in vivo. Neutrophils entering post-capillary venules adjacent to inflammatory foci develop transient rolling adhesive interactions with endothelium via selectins (6). Following exposure to inflammatory cytokines such as tumor necrosis factor and interleukin-1, endothelial cells are induced to express E-selectin and P-selectin (6). Several surface glycoproteins on neutrophils, including L-selectin and P-selectin glycoprotein ligand 1, present oligosaccharide moieties that serve as counter receptors for E-selectin and P-selectin. In conjunction with neutrophil membrane L-selectin, which recognizes oligosaccharides on endothelial cells,

Macrophage migration inhibitory factor (MIF) is a cytokine that was first described as an inhibitor of the random migration of monocytes and macrophages and has since been proposed to have a number of immune and catalytic functions. One of the functions assigned to MIF is that of a tautomerase that interconverts the enol and keto forms of phenylpyruvate and (p-hydroxyphenyl)pyruvate and converts D-dopachrome, a stereoisomer of naturally occurring L-dopachrome, to 5,6-dihydroxyindole-2-carboxylic acid. The physiological significance of the MIF enzymatic activity is unclear. The three-dimensional structure of MIF is strikingly similar to that of two microbial enzymes (4-oxalocrotonate tautomerase and 5-carboxymethyl-2-hydroxymuconate isomerase) that otherwise share little sequence identity with MIF. MIF and these two enzymes have an invariant N-terminal proline that serves as a catalytic base. Here we report a new biological function for MIF, as an inhibitor of monocyte chemoattractant protein 1- (MCP-1-) induced chemotaxis of human peripheral blood monocytes. We find that MIF inhibition of chemotaxis does not occur at the level of the CC chemokine receptor for MCP-1, CCR2, since MIF does not alter the binding of (125)I-MCP-1 to monocytes. The role of MIF enzymatic activity in inhibition of monocyte chemotaxis and random migration was studied with two MIF mutants in which the N-terminal proline was replaced with either a serine or a phenylalanine. Both mutants remain capable of inhibiting monocyte chemotaxis and random migration despite significantly reduced or no phenylpyruvate tautomerase activity. These data suggest that this enzymatic activity of MIF does not play a role in its migration inhibiting properties.

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SummaryThe Myxococcus xanthus gene, pkn9, encodes a protein that contains significant homology with eukaryotic Ser/ Thr protein kinases. The pkn9 gene was singled out of a previously identified family of kinase genes by amplification techniques that displayed differences in kinase gene expression during selected periods of the M. xanthus life cycle. Pkn9 was constitutively expressed during vegetative growth and upregulated during the aggregation stage of early development. It consists of 589 amino acids, and its N-terminal 394 residues show 38% identity with both Pkn1 and Pkn2 of M. xanthus. This region also shows 29, 25 and 29% identity with myosin light-chain kinase, protein kinase C, and cAMP-dependent protein kinase, respectively. A 22-residue hydrophobic transmembrane domain separates the kinase domain from the 173-residue C-terminal domain that resides on the outside of the inner membrane. The C-terminal domain contains two sets of tandem repeats of 13 and 10 residues which have no known function. When expressed in Escherichia coli under the T7 promoter, Pkn9 was found to be phosphorylated on serine and threonine residues. Disruption of the pkn9 kinase catalytic subdomains I-III by the insertion of a kanamycin-resistance gene resulted in slightly delayed, smaller and more-crowded fruiting bodies, while spore formation was normal. Total deletion of the pkn9 gene caused severely reduced progression through development resulting in light loose mounds that become slightly more compact over time. Development progressed further at the centre than at the edge of the spot, and spore formation was significantly reduced. Twodimensional gel analysis revealed that both the disruption and the deletion of pkn9 prevented the expression of five membrane proteins (KREP9-1-4). These results suggest that the loss of Pkn9 kinase activity caused altered fruiting-body formation, the absence of the KREP9 proteins in the membrane, and reduced spore production.

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