Purification of MAP-kinase protein complexes and identification of candidate components by XL-TAP-MS.

The purification of low-abundance protein complexes and detection of in vivo protein-protein interactions in complex biological samples remains a challenging task. Here, we devised crosslinking and tandem affinity purification coupled to mass spectrometry (XL-TAP-MS), a quantitative proteomics approach for analyzing tandem affinity-purified, crosslinked protein complexes from plant tissues. We exemplarily applied XL-TAP-MS to study the MKK2-Mitogen-activated protein kinase (MPK4) signaling module in Arabidopsis thaliana. A tandem affinity tag consisting of an in vivo-biotinylated protein domain flanked by two hexahistidine sequences was adopted to allow for the affinity-based isolation of formaldehyde-crosslinked protein complexes under fully denaturing conditions. Combined with 15N stable isotopic labeling and tandem MS we captured and identified a total of 107 MKK2-MPK4 module-interacting proteins. Consistent with the role of the MPK signaling module in plant immunity, many of the module-interacting proteins are involved in the biotic and abiotic stress response of Arabidopsis. Validation of binary protein-protein interactions by in planta split-luciferase assays and in vitro kinase assays disclosed several direct phosphorylation targets of MPK4. Together, the XL-TAP-MS approach purifies low abundance protein complexes from biological samples and discovers previously unknown protein-protein interactions.

Detection and Functional Analysis of SUMO-Modified MEK.

Small ubiquitin-like modifier (SUMO) is a posttranslational protein modifier that binds target proteins covalently (protein sumoylation) and remarkably alters their functions. Protein sumoylation has been linked to various cellular functions such as cell division, DNA repair, and import of nuclear proteins. Thus, its dysregulation is implicated in diverse human diseases such as neurodegenerative disorders and cancers. We recently found that the kinase activity of MEK proteins, which function as central components of the ERK-MAPK cascade and amplify an extracellular proliferation signal, is negatively regulated by sumoylation. Moreover, the oncogenic activity of Ras is enhanced by the abrogation of MEK-sumoylation in cancer cells. Here, we describe several tools and techniques utilized for the elucidation of the properties of SUMO-MEK in our previous reports. We believe that these methods can be used as robust tools for investigating and understanding the biological roles of various SUMO-modified (sumoylated) proteins.

Production of active recombinant mitogen-activated protein kinases through transient transfection of 293T cells.

Mitogen-activated protein (MAP) kinases are a family of serine/threonine protein kinases that play an important role in a myriad of cellular processes, including cell proliferation, differentiation, and apoptosis. Abnormal activation of MAP kinases has been shown to participate in a variety of human diseases which include cancer, septic shock, rheumatoid arthritis, diabetes, and cardiovascular diseases. Active MAP kinase enzymes are not only valuable for basic biomedical research but are also critical for the development of pharmacological inhibitors as therapeutic drugs in the treatment of relevant human diseases. MAP kinases produced in a bacterial system are poorly active due to a lack of proper phosphorylation at their characteristic threonine and tyrosine residues. To overcome these limitations, we have developed a mammalian expression system for high level expression and one-step purification of enzymatically MAP kinases. We cloned JNK1, p38, and p38-regulated MAP kinase-activated protein kinase-2 into the mammalian expression vector pEBG, and expressed these protein kinases as glutathione S-transferase fusion proteins in human embryonic kidney 293T cells through transient transfection. The protein kinases were activated in vivo through treating the transfected cells with sodium arsenite and affinity-purified using glutathione-Sepharose beads. The enzymatic activities of these protein kinases were demonstrated by Western blot analysis and in vitro kinase assays. Our results indicate that this system is an extremely powerful tool for generating valuable reagents, and could be very valuable for proteomic studies.

LmxPK4, a mitogen-activated protein kinase kinase homologue of Leishmania mexicana with a potential role in parasite differentiation.

Members of the mitogen-activated protein (MAP) kinase cascade are important for the establishment of a Leishmania mexicana infection and are involved in flagellar length control, although the underlying molecular mechanisms remain to be elucidated. This study reports the cloning and characterization of LmxPK4, a MAP kinase kinase homologue of L. mexicana displaying putative plant-like regulatory phosphorylation sites. The recombinant protein has autophosphorylating activity and phosphorylates myelin basic protein. An LmxPK4 gene deletion mutant showed a proliferation defect after infection of macrophages and no or delayed lesion development in mice. Irrespective of the onset of lesion development parasites showed an early and homogeneous lesion development in re-infection experiments. This is indicative for a compensation of the null mutant phenotype. Additionally, this phenotype could be reverted by reintroduction of the wild-type gene into the deletion background. Mutants expressing loss-of-function or N-terminally truncated versions of LmxPK4 retained the null mutant phenotype. LmxPK4 is stage-specifically expressed in promastigotes and during differentiation to amastigotes, but is not detectable in amastigotes isolated from the mammalian host. Moreover, its in vitro kinase activity increases with temperature rise up to 40 degrees C. Our results suggest that LmxPK4 is involved in the differentiation process and affects virulence of Leishmania mexicana.

The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis.

The Arabidopsis mitogen-activated protein kinase (MAPK) kinase 2 (MKK2) and the downstream MAPKs MPK4 and MPK6 were isolated by functional complementation of osmosensitive yeast mutants. In Arabidopsis protoplasts, MKK2 was specifically activated by cold and salt stress and by the stress-induced MAPK kinase kinase MEKK1. Yeast two-hybrid, in vitro, and in vivo protein kinase assays revealed that MKK2 directly targets MPK4 and MPK6. Accordingly, plants overexpressing MKK2 exhibited constitutive MPK4 and MPK6 activity, constitutively upregulated expression of stress-induced marker genes, and increased freezing and salt tolerance. In contrast, mkk2 null plants were impaired in MPK4 and MPK6 activation and were hypersensitive to salt and cold stress. Full genome transcriptome analysis of MKK2-overexpressing plants demonstrated altered expression of 152 genes involved in transcriptional regulation, signal transduction, cellular defense, and stress metabolism. These data identify a MAP kinase signaling cascade mediating cold and salt stress tolerance in plants.

Protein kinase involved in flagellar-length control.

During its life cycle, the parasitic protozoon Leishmania mexicana differentiates from a flagellated form, the promastigote, to an amastigote form carrying a rudimentary flagellum. Besides biochemical changes, this process involves a change in overall cell morphology including flagellar shortening. A mitogen-activated protein kinase kinase homologue designated LmxMKK was identified in a homology screening and found to be critically involved in the regulation of flagellar assembly and cell size. LmxMKK is exclusively expressed in the promastigote stage and is likely to be regulated by posttranslational mechanisms such as phosphorylation. A deletion mutant for the single-copy gene revealed motile flagella dramatically reduced in length and lacking the paraflagellar rod, a structure adjacent to the axoneme in kinetoplastid flagella. Moreover, a fraction of the cells showed perturbance of the axonemal structure. Complementation of the deletion mutant with the wild-type gene restored typical promastigote morphology. We propose that LmxMKK influences anterograde intraflagellar transport to maintain flagellar length in Leishmania promastigotes; as such, it is the first protein kinase known to be involved in organellar assembly.

Regulation of meiotic progression by the meiosis-specific checkpoint kinase Mek1 in fission yeast.

During the eukaryotic cell cycle, accurate transmission of genetic information to progeny is ensured by the operation of cell cycle checkpoints. Checkpoints are regulatory mechanisms that block cell cycle progression when key cellular processes are defective or chromosomes are damaged. During meiosis, genetic recombination between homologous chromosomes is essential for proper chromosome segregation at the first meiotic division. In response to incomplete recombination, the pachytene checkpoint (also known as the meiotic recombination checkpoint) arrests or delays meiotic cell cycle progression, thus preventing the formation of defective gametes. Here, we describe a role for a meiosis-specific kinase, Mek1, in the meiotic recombination checkpoint in fission yeast. Mek1 belongs to the Cds1/Rad53/Chk2 family of kinases containing forkhead-associated domains, which participate in a number of checkpoint responses from yeast to mammals. We show that defects in meiotic recombination generated by the lack of the fission yeast Meu13 protein lead to a delay in entry into meiosis I owing to inhibitory phosphorylation of the cyclin-dependent kinase Cdc2 on tyrosine 15. Mutation of mek1(+) alleviates this checkpoint-induced delay, resulting in the formation of largely inviable meiotic products. Experiments involving ectopic overexpression of the mek1(+) gene indicate that Mek1 inhibits the Cdc25 phosphatase, which is responsible for dephosphorylation of Cdc2 on tyrosine 15. Furthermore, the meiotic recombination checkpoint is impaired in a cdc25 phosphorylation site mutant. Thus, we provide the first evidence of a connection between an effector kinase of the meiotic recombination checkpoint and a crucial cell cycle regulator and present a model for the operation of this meiotic checkpoint in fission yeast.

Convergence and divergence of stress-induced mitogen-activated protein kinase signaling pathways at the level of two distinct mitogen-activated protein kinase kinases.

Plants respond to biotic and abiotic stresses by inducing overlapping sets of mitogen-activated protein kinases (MAPKs) and response genes. To define the mechanisms of how different signals can activate a common signaling pathway, upstream activators of SIMK, a salt stress- and pathogen-induced alfalfa MAPK, were identified. Here, we compare the properties of SIMKK, a MAPK kinase (MAPKK) that mediates the activation of SIMK by salt stress, with those of PRKK, a distantly related novel MAPKK. Although both SIMKK and PRKK show strongest interaction with SIMK, SIMKK can activate SIMK without stimulation by upstream factors. In contrast, PRKK requires activation by an upstream activated MAPKK kinase. SIMKK mediates pathogen elicitor signaling and salt stress, but PRKK transmits only elicitor-induced MAPK activation. Of four tested MAPKs, PRKK activates three of them (SIMK, MMK3, and SAMK) upon elicitor treatment of cells. However, PRKK is unable to activate any MAPK upon salt stress. In contrast, SIMKK activates SIMK and MMK3 in response to elicitor, but it activates only SIMK upon salt stress. These data show that (1) MAPKKs function as convergence points for stress signals, (2) MAPKKs activate multiple MAPKs, and (3) signaling specificity is obtained not only through the inherent affinities of MAPKK-MAPK combinations but also through stress signal-dependent intracellular mechanisms.

ATMPK4, an Arabidopsis homolog of mitogen-activated protein kinase, is activated in vitro by AtMEK1 through threonine phosphorylation.

The modulation of mitogen-activated protein kinase (MAPK) activity regulates many intracellular signaling processes. In animal and yeast cells, MAP kinases are activated via phosphorylation by the dual-specificity kinase MEK (MAP kinase kinase). Several plant homologs of MEK and MAPK have been identified, but the biochemical events underlying the activation of plant MAPKs remain unknown. We describe the in vitro activation of an Arabidopsis homolog of MAP kinase, ATMPK4. ATMPK4 was phosphorylated in vitro by an Arabidopsis MEK homolog, AtMEK1. This phosphorylation occurred principally on threonine (Thr) residues and resulted in elevated ATMPK4 kinase activity. A second Arabidopsis MEK isoform, ATMAP2Kalpha, failed to phosphorylate ATMPK4 in vitro. Tyr dephosphorylation by the Arabidopsis Tyr-specific phosphatase AtPTP1 resulted in an almost complete loss of ATMPK4 activity. Immunoprecipitates of Arabidopsis extracts with anti-ATMPK4 antibodies displayed myelin basic protein kinase activity that was sensitive to treatment with AtPTP1. These results demonstrate that a plant MEK can phosphorylate and activate MAPK, and that Tyr phosphorylation is critical for the catalytic activity of MAPK in plants. Surprisingly, in contrast to the animal enzymes, AtMEK1 may not be a dual-specificity kinase but, rather, the required Tyr phosphorylation on ATMPK4 may result from autophosphorylation.