Mitogen-activated protein kinase Mkp1 of Pneumocystis carinii complements the slt2Delta defect in the cell integrity pathway of Saccharomyces cerevisiae

Signal transduction pathways are important in the adaptive response of microbes to their environment. A Pneumocystis carinii extracellular signal-regulated protein kinase (MAPK) homologue, Mkp1, has been isolated by sequence similarity screening of P. carinii genomic DNA. The Mkp1 of P. carinii shows closest homology to other fungal MAP kinases involved in cell integrity signal transduction cascades, including Slt2p/Mpk1p of Saccharomyces cerevisiae, Mkc1 of Candida albicans and Mps1 of Magnaporthe grisea. Defects of Slt2p in S. cerevisiae result in phenotypes of slow growth, and temperature sensitivity in the absence of an osmostabilizer. Overexpression of mkp1 in a strain with the slt2Delta defect fully restored the normal growth rate, and partially reduced lysis at elevated temperatures. Complementation of the slt2Delta defect by Mkp1 demonstrates that Mkp1 is a functional MAP kinase, and that it may be the MAP kinase component of a similar signal transduction cascade within P. carinii. Furthermore, Mkp1 is activated in vitro upon the exposure of P. carinii to conditions of oxidative stress. The investigation of a MAP kinase signal transduction pathway of P. carinii will result in both a better understanding of the mechanism the organism utilizes to respond to environmental changes, and a system to assay responses to these changes.     

Characterization of domains in the yeast MAP kinase Slt2 (Mpk1) required for functional activity and in vivo interaction with protein kinases Mkk1 and Mkk2

MKK1/MKK2 and SLT2 ( MPK1 ) are three Saccharomyces cerevisiae genes, coding for protein kinases, that have been postulated to act sequentially as part of the Pkc1p signalling pathway, a phosphorylation cascade essential for cell integrity. By using the 'two-hybrid system' and co-purification experiments on glutathione-agarose beads, we have shown that Slt2p interacts in vivo and in vitro with both Mkk1p and Mkk2p, thus confirming a previous suggestion based on epistasis experiments of the corresponding genes. Plasmid constructs of the SLT2 gene, deleted in the whole C-terminal non-kinase region or part of it, and therefore containing all of the conserved kinase subdomains, were still functional in complementation of the slt2 lytic phenotype and in vivo interaction with Mkk1p and Mkk2p. In contrast, the Slt2p C-terminal domain (162 residues) that carries a glutamine-rich fragment followed by a 16 polyglutamine tract, was shown to be dispensable for complementation and in vivo association with Mkk1p and Mkk2p. We have also demonstrated that the N-terminal putative regulatory domain of these two MAP kinase activators is the main region involved in the interaction with Slt2p.     

Quantification of the Dynamic Phosphorylation Process of ERK Using Stable Isotope Dilution Selective Reaction Monitoring Mass Spectrometry

Mitogen‐activated protein (MAP) kinase signaling is critical for various cellular responses, including cell proliferation, differentiation, and cell death. The MAP kinase cascade is conserved in the eukaryotic kingdom as a three‐tiered kinase module—MAP kinase kinase kinase, MAP kinase kinase, and MAP kinase—that transduces signals via sequential phosphorylation upon stimulation. Dual phosphorylation of MAP kinase on the conserved threonine‐glutamic acid‐tyrosine (TEY) motif is essential for its catalytic activity and signal activation; however, the molecular mechanism by which the two residues are phosphorylated remains elusive. In the present study, the pattern of dual phosphorylation of extracellular signal‐regulated kinase (ERK) is profiled on the TEY motif using stable isotope dilution (SID)‐selective reaction monitoring (SRM) mass spectrometry (MS) to elucidate the order and magnitude of endogenous ERK phosphorylation in cellular model systems. The SID‐SRM‐MS analysis of phosphopeptides demonstrates that tyrosine phosphorylation in the TEY motif is dynamic, while threonine phosphorylation is static. Analyses of the mono‐phosphorylatable mutants ERK^T202A and ERK^Y204F indicate that phosphorylation of tyrosine is not affected by the phosphorylation state of threonine, while threonine phosphorylation depends on tyrosine phosphorylation. The data suggest that dual phosphorylation of ERK is a highly ordered and restricted mechanism determined by tyrosine phosphorylation.     

Activation loop phosphorylation of the atypical MAP kinases ERK3 and ERK4 is required for binding, activation and cytoplasmic relocalization of MK5

Mitogen-activated protein (MAP) kinases are typical examples of protein kinases whose enzymatic activity is mainly controlled by activation loop phosphorylation. The classical MAP kinases ERK1/ERK2, JNK, p38 and ERK5 all contain the conserved Thr-Xxx-Tyr motif in their activation loop that is dually phosphorylated by members of the MAP kinase kinases family. Much less is known about the regulation of the atypical MAP kinases ERK3 and ERK4. These kinases display structural features that distinguish them from other MAP kinases, notably the presence of a single phospho-acceptor site (Ser-Glu-Gly) in the activation loop. Here, we show that ERK3 and ERK4 are phosphorylated in their activation loop in vivo. This phosphorylation is exerted, at least in part, in trans by an upstream cellular kinase. Contrary to classical MAP kinases, activation loop phosphorylation of ERK3 and ERK4 is detected in resting cells and is not further stimulated by strong mitogenic or stress stimuli. However, phosphorylation can be modulated indirectly by interaction with the substrate MAP kinase-activated protein kinase 5 (MK5). Importantly, we found that activation loop phosphorylation of ERK3 and ERK4 stimulates their intrinsic catalytic activity and is required for the formation of stable active complexes with MK5 and, consequently, for efficient cytoplasmic redistribution of ERK3/ERK4-MK5 complexes. Our results demonstrate the importance of activation loop phosphorylation in the regulation of ERK3/ERK4 function and highlight differences in the regulation of atypical MAP kinases as compared to classical family members.