ABSTRACT
Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) is activated upon stress by p38 MAPK alpha and -beta, which bind to a basic docking motif in the C terminus of MK2 and which subsequently phosphorylate its regulatory sites. As a result of activation MK2 is exported from the nucleus to the cytoplasm and cotransports active p38 MAPK to this compartment. Here we show that the amount of p38 MAPK is significantly reduced in cells and tissues lacking MK2, indicating a stabilizing effect of MK2 for p38. Using a murine knockout model, we have previously shown that elimination of MK2 leads to a dramatic reduction of tumor necrosis factor (TNF) production in response to lipopolysaccharide. To further elucidate the role of MK2 in p38 MAPK stabilization and in TNF biosynthesis, we analyzed the ability of two MK2 isoforms and several MK2 mutants to restore both p38 MAPK protein levels and TNF biosynthesis in macrophages. We show that MK2 stabilizes p38 MAPK through its C terminus and that MK2 catalytic activity does not contribute to this stabilization. Importantly, we demonstrate that stabilizing p38 MAPK does not restore TNF biosynthesis. TNF biosynthesis is only restored with MK2 catalytic activity. We further show that, in MK2-deficient macrophages, formation of filopodia in response to extracellular stimuli is reduced. In addition, migration of MK2-deficient mouse embryonic fibroblasts (MEFs) and smooth muscle cells on fibronectin is dramatically reduced. Interestingly, reintroducing catalytic MK2 activity into MEFs alone is not sufficient to revert the migratory phenotype of these cells. In addition to catalytic activity, the proline-rich N-terminal region is necessary for rescuing the migratory phenotype. These data indicate that catalytic activity of MK2 is required for both cytokine production and cell migration. However, the proline-rich MK2 N terminus provides a distinct role restricted to cell migration.
Subject(s)
Macrophages/metabolism , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Protein Kinases , Adenoviridae/genetics , Amino Acid Motifs , Amino Acid Sequence , Animals , Cell Movement/genetics , Cells, Cultured , Fibroblasts , Intracellular Signaling Peptides and Proteins , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Mice , Mice, Mutant Strains , Molecular Sequence Data , Muscle, Smooth/cytology , Mutation , Nuclear Localization Signals/genetics , Nuclear Localization Signals/metabolism , Organ Specificity , Proline , Protein Serine-Threonine Kinases , Protein Structure, Tertiary , Tumor Necrosis Factor-alpha/metabolism , p38 Mitogen-Activated Protein KinasesABSTRACT
Three mitogen activated protein kinase-activated protein kinase 2 (MAPKAP kinase 2, MK2) interacting proteins were identified using a yeast two-hybrid approach. ShcA, a signaling phospho-protein, human polyhomeotic 2 (HPH2), a transcriptional regulator, and highly similar to smoothelin (HSTS), which is related to the cytoskeletal associated protein smoothelin, interact specifically with MK2. The interaction of MK2 with the 66 kDa isoform of ShcA, p66(ShcA), and HPH2 was confirmed using co-immunoprecipitation. MK2 is activated with p66(ShcA) co-expression and p66(ShcA) is an in vitro substrate for MK2, further demonstrating their association and suggesting a biological role for p66(Shc) in MK2 activation.