Protein interactions between MAP kinases and substrates, activators, and scaffolding proteins are regulated by docking site motifs, one containing basic residues proximal to Leu-X-Leu (DEJL) and a second containing Phe-X-Phe (DEF). Hydrogen exchange mass spectrometry was used to identify regions in MAP kinases protected from solvent by docking motif interactions. Protection by DEJL peptide binding was observed in loops spanning beta7-beta8 and alphaD-alphaE in p38alpha and ERK2. In contrast, protection by DEF binding to ERK2 revealed a distinct hydrophobic pocket for Phe-X-Phe binding formed between the P+1 site, alphaF helix, and the MAP kinase insert. In inactive ERK2, this pocket is occluded by intramolecular interactions with residues in the activation lip. In vitro assays confirm the dependence of Elk1 and nucleoporin binding on ERK2 phosphorylation, and provide a structural basis for preferential involvement of active ERK in substrate binding and nuclear pore protein interactions.
We recently cloned a novel signaling molecule, p122, that shows a GTPase-activating activity specific for Rho and the ability to enhance the phosphatidylinositol 4,5-bisphosphate-hydrolyzing activity of phospholipase C ␦1 in vitro. Here we analyzed the in vivo function of p122. Microinjection of the GTPase-activating domain of p122 suppressed the formation of stress fibers and focal adhesions induced by lysophosphatidic acid, suggesting a GTPase-activating activity for Rho as in in vitro. Transfection of p122 also induced the disassembly of stress fibers and the morphological rounding of various adherent cells. Analyses using deletion and point mutants demonstrated that the GTPase-activating domain of p122 is responsible for the morphological changes and detachment and that arginine residues at positions 668 and 710 and a lysine residue at position 706 in the GTPase-activating domain are essential. Using Fluo-3-based Ca 2؉ microscopy, we found that p122 evoked a rapid elevation of intracellular Ca 2؉ levels, suggesting that p122 stimulates the phosphatidylinositol 4,5-bisphosphate-hydrolyzing activity of phospholipase C ␦1. These results demonstrate that p122 synergistically functions as a GTPase-activating protein specific for Rho and an activator of phospholipase C ␦1 in vivo and induces morphological changes and detachment through cytoskeletal reorganization.Recent studies have indicated a close association between the regulation of cytoskeletal assembly and phosphatidylinositol (PI) 1 metabolism. That is, a number of PI 4,5-bisphosphate (PIP 2 )-binding proteins including gelsolin, cofilin, profilin, and ␣-actinin are known to bind to actin and regulate cytoskeletal assembly (1-4). In addition, Rho has been shown to enhance the activity of PI 4-phosphate 5-kinase, the PIP 2 -synthesizing enzyme (5), and the overexpression of PI 4-phosphate 5-kinase induces massive actin polymerization in COS-7 cells (6). Alternatively, microinjection of antibodies against PIP 2 inhibits the formation of stress fibers and focal adhesions (7). These results strongly suggest that PIP 2 newly synthesized by PI 4-phosphate 5-kinase, which exists downstream of Rho, binds to PIP 2 -binding proteins (i.e. actin-binding protein) to release free Gactins.These reactions increase the intracellular concentrations of free G-actins, resulting in their reorganization to form actin fibers (8).On the other hand, Rho is inactivated by GTPase-activating proteins (GAPs) (9 -12). It is possible that Rho GAPs, as downstream components of Rho, regulate cytoskeletal disassembly. We recently cloned a GAP as a novel molecule that interacts with phospholipase C ␦1 (PLC␦1) (13). In addition to the GAP activity specific for Rho in vitro, p122 possesses the ability to enhance the PIP 2 -hydrolyzing activity of PLC␦1 in vitro. Therefore, it is possible that p122 is an ideal link between Rho and cytoskeletal disassembly. To address this issue, we tried to isolate cells stably overexpressing p122. However, we have never succeeded in obtaining such cells, sug...
Abnormal amplification of centrosomes, commonly found in human cancer, is the major cause of mitotic defects and chromosome instability in cancer cells. Like DNA, centrosomes duplicate once in each cell cycle, hence the defect in the mechanism that ensures centrosome duplication to occur once and only once in each cell cycle results in abnormal amplification of centrosomes and mitotic defects. Centrosomes are non-membranous organelles, and undergo dynamic changes in its constituents during the centrosome duplication cycle. Through a comparative mass spectrometric analysis of unduplicated and duplicated centrosomes, we identified mortalin, a member of heat shock protein family, as a protein that associates preferentially with duplicated centrosomes. Further analysis revealed that mortalin localized to centrosomes in late G1 before centrosome duplication, remained at centrosomes during S and G2, and dissociated from centrosomes during mitosis. Overexpression of mortalin overrides the p53-dependent suppression of centrosome duplication, and mortalin-driven centrosome duplication requires physical interaction between mortalin and p53. Moreover, mortalin promotes dissociation of p53 from centrosomes through physical interaction. The p53 mutant that lacks the ability to bind to mortalin remains at centrosomes, and suppresses centrosome duplication in a transactivation function-independent manner. Thus, our present findings not only identify mortalin as an upstream molecule of p53 but also provide evidence for the involvement of centrosomally localized p53 in the regulation of centrosome duplication.
The biological role of phosphatidylinositol (PI)-3 kinase was examined in osteoclast-like multinucleated cells (OCLs) formed in co-cultures of mouse osteoblastic ceils and bone marrow cells. The expression of PI-3 kinase in OCLs was confirmed by Western blot analysis. Wortmannin (WT), a specific inhibitor of PI-3 kinase, inhibited PI-3 kinase activity in OCLs both in vitro and in vivo. WT also inhibited pit-forming activity on dentine slices and disrupted a ringed structure of F-actin-containing dots (an actin ring) in OCLs in a dose-dependent manner. The inhibitory profiles of WT for pit and actin ring formation were similar to that for PI-3 kinase activity in OCLs. Electron microscopic analysis revealed that OCLs treated with WT did not form ruffled borders. Instead, numerous electron lucent vacuoles of differing sizes were found throughout the cytoplasm. These results suggest that PI-3 kinase is important in osteoclastic bone resorption.
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