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Immediate early gene (IEG) expression is coordinated by multiple MAP kinase signaling pathways in a signal specific manner. Stress-activated p38α MAP kinase is implicated in transcriptional regulation of IEGs via MSK-mediated CREB phosphorylation. The protein kinases downstream to p38, MAPKAP kinase (MK) 2 and MK3 have been identified to regulate gene expression at the posttranscriptional levels of mRNA stability and translation. Here, we analyzed stress-induced IEG expression in MK2/3-deficient cells. Ablation of MKs causes a decrease of p38α level and p38-dependent IEG expression. Unexpectedly, restoration of p38α does not rescue the full-range IEG response. Instead, the catalytic activity of MKs is necessary for the major transcriptional activation of IEGs. By transcriptomics, we identified MK2-regulated genes and recognized the serum response element (SRE) as a common promoter element. We show that stress-induced phosphorylation of serum response factor (SRF) at serine residue 103 is significantly reduced and that induction of SRE-dependent reporter activity is impaired and can only be rescued by catalytically active MK2 in MK2/3-deficient cells. Hence, a new function of MKs in transcriptional activation of IEGs via the p38α-MK2/3-SRF-axis is proposed which probably cooperates with MKs’ role in posttranscriptional gene expression in inflammation and stress response.

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The scratch wound healing assay is a sensitive method to characterize cell proliferation and migration, but it is difficult to be quantitatively evaluated. Therefore, we developed an infrared fluorescence detection-based real-time assay for sensitive and accurate quantification of cell migration in vitro. The method offers sensitivity, simplicity, and the potential for integration into automated large-scale screening studies. A live cell staining lipophilic tracer-1,1 0 -dioctadecyl-3,3,3 0 ,3 0 -tetramethyl indotricarbocyanine iodide (DiR)-is used for accurate imaging of wound closure in a simple 96-well scratch assay. Scratches are made on prestained confluent cell monolayers using a pipette tip and scanned at different time intervals using a fluorescent scanner. Images are analyzed using Image J software and the migration index is calculated. Effect of cell number, time after scratch and software settings are analyzed. The method is validated by showing concentration-and time-dependent effects of cytochalasin-D on fibroblast migration. Using this assay, we quantitatively evaluate the role of the MAPK-activated protein kinases MK2 and MK3 in fibroblast migration. First, the migratory phenotype of MK2-deficient MEFs is analyzed in a retroviral rescue model. In addition, migration of MK2/3-double-deficient cells is determined and the ability of MK3 to rescue cell migration in MK2/3-double-deficient fibroblasts is demonstrated.

The MAPK-activated protein kinases (MAPKAP kinases) MK2 and MK3 are directly activated via p38 MAPK phosphorylation, stabilize p38 by complex formation, and contribute to the stress response. The list of substrates of MK2/3 is increasing steadily. We applied a phosphoproteomics approach to compare protein phosphorylation in MK2/3-deficient cells rescued or not by ectopic expression of MK2. In addition to differences in phosphorylation of the known substrates of MK2, HSPB1 and Bag-2, we identified strong differences in phosphorylation of keratin 8 (K8 Physiologic relevance of these findings was confirmed by differences of K20-Ser 13 phosphorylation between the ileum of wildtype and MK2/3-deficient mice and by demonstrating p38-and MK2-dependent mucin secretion of HT29 cells. Therefore, MK2 and p38 MAPK function in concert to phosphorylate K8, K18, and K20 in intestinal epithelia.Intermediate filaments are important structural and functional components of eukaryotic cells. Together with the microfilament and microtubule network they constitute the third major filament system of the cytoskeleton, characterized by their unique expression patterns and nonpolar filament association. Keratins (K) 2 are cytoplasmic intermediate filaments preferentially expressed in the epithelium. The 54 keratin genes in human genome can be divided into type I (acidic) and type II (basic) keratins. They exist as obligate noncovalent heteropolymers consisting of at least one type I (K9 -K28, K31-40) and one type II keratin (K1-K8, K71-86) in an equimolar ratio (1). The keratins of the single layered epithelium mainly express K8 and K18 and, depending on the cell type, variable amounts of K7, K19, and K20 (2). For example, hepatocytes exclusively express K8/18, whereas intestinal epithelial cells express K8/18/19/20. Several post-translational modifications are known to modulate the keratin filaments. Phosphorylation is by far the most studied modification of keratins, and phosphorylation sites were identified in all major keratins of the single-layered epithelium (K8/18/19/20) (3).The p38 MAPK (p38) pathway is activated by proinflammatory cytokines, radiation, and osmotic and chemical stress (4). p38 has been implicated as the physiological protein kinase responsible for K8 phosphorylation during mitosis and under various cellular stress stimuli (5). Pharmacological inhibition of p38 using SB202190 was shown to be sufficient to block orthovanadate-induced keratin filament breakdown completely (6). K8-Ser 73 phosphorylation by p38 was associated with, but not sufficient for keratin filament destabilization. This suggested involvement of additional p38-dependent keratin phosphorylation sites.MAPK-activating protein (MAPKAP) kinases 2 (MK2) and 3 (MK3) are well characterized downstream targets of p38, with MK2 having predominant physiological relevance due to its higher abundance compared with MK3 (7). Two distinct roles for MK2 in the cellular stress response involve cytokine biosynthesis and cytoskeleton modulation. MK2/3 are shown to p...