In vitro activity characterization of the tomato SnRK1 complex proteins.

Plant Sucrose non-Fermenting 1-Related Protein Kinase1 (SnRK1) complexes are members of the Snf1/AMPK/SnRK protein kinase family and play important roles in many aspects of metabolism. In tomato (Solanum lycopersicum, Sl), only one α-subunit of the SnRK1 complex, SlSnRK1.1, has been characterized to date. In this study, the phylogenetic placement and in vitro kinase activity of a second tomato SnRK1 α-subunit, SlSnRK1.2, were characterized. Interestingly, in the phylogenetic analysis of SnRK1 sequences from monocots and dicots SlSnRK1.2 clusters only with other Solanaceae SnRK1.2 sequences, suggesting possible functional divergence of these kinases from other SnRK1 kinases. For analysis of kinase activity, SlSnRK1.2 was able to autophosphorylate, phosphorylate the complex ß-subunits, and phosphorylate the SnRK1 AMARA peptide substrate, all with drastically lower overall kinase activity compared to SlSnRK1.1. Activation by the upstream kinase SlSnAK was able to increase the kinase activity of both SlSnRK1.1 and SlSnRK1.2, although the increase is less dramatic for SlSnRK1.2. The highest kinase activity on the AMARA peptide for SlSnRK1.2 was seen when reconstituting the complex in vitro with SlSip1 as the ß-subunit. In comparison, SlSnRK1.1 showed the lowest kinase activity on the AMARA peptide when SlSip1 was used. These studies suggest the SlSnRK1.2 phylogenetic divergence and lower SlSnRK1.2 kinase activity compared to SlSnRK1.1 may be indicative of different in vivo roles for each kinase.

The tomato cell death suppressor Adi3 is restricted to the endosomal system in response to the Pseudomonas syringae effector protein AvrPto.

The tomato (Solanum lycopersicum) AGC protein kinase Adi3 functions as a suppressor of cell death and was first identified as an interactor with the tomato resistance protein Pto and the Pseudomonas syringae effector protein AvrPto. Models predict that loss of Adi3 cell death suppression (CDS) activity during Pto/AvrPto interaction leads to the cell death associated with the resistance response initiated from this interaction. Nuclear localization is required for Adi3 CDS. Prevention of nuclear accumulation eliminates Adi3 CDS and induces cell death by localizing Adi3 to intracellular punctate membrane structures. Here we use several markers of the endomembrane system to show that the punctate membrane structures to which non-nuclear Adi3 is localized are endosomal in nature. Wild-type Adi3 also localizes in these punctate endosomal structures. This was confirmed by the use of endosomal trafficking inhibitors, which were capable of trapping wild-type Adi3 in endosomal-like structures similar to the non-nuclear Adi3. This suggests Adi3 may traffic through the cell using the endomembrane system. Additionally, Adi3 was no longer found in the nucleus but was visualized in these punctate endosomal-like membranes during the cell death induced by the Pto/AvrPto interaction. Therefore we propose that inhibiting nuclear import and constraining Adi3 to the endosomal system in response to AvrPto is a mechanism to initiate the cell death associated with resistance.

The ß-subunit of the SnRK1 complex is phosphorylated by the plant cell death suppressor Adi3.

The protein kinase AvrPto-dependent Pto-interacting protein3 (Adi3) is a known suppressor of cell death, and loss of its function has been correlated with cell death induction during the tomato (Solanum lycopersicum) resistance response to its pathogen Pseudomonas syringae pv tomato. However, Adi3 downstream interactors that may play a role in cell death regulation have not been identified. We used a yeast two-hybrid screen to identify the plant SnRK1 (for Sucrose non-Fermenting-1-Related Protein Kinase1) protein as an Adi3-interacting protein. SnRK1 functions as a regulator of carbon metabolism and responses to biotic and abiotic stresses. SnRK1 exists in a heterotrimeric complex with a catalytic α-subunit (SnRK1), a substrate-interacting ß-subunit, and a regulatory γ-subunit. Here, we show that Adi3 interacts with, but does not phosphorylate, the SnRK1 α-subunit. The ability of Adi3 to phosphorylate the four identified tomato ß-subunits was also examined, and it was found that only the Galactose Metabolism83 (Gal83) ß-subunit was phosphorylated by Adi3. This phosphorylation site on Gal83 was identified as serine-26 using a mutational approach and mass spectrometry. In vivo expression of Gal83 indicates that it contains multiple phosphorylation sites, one of which is serine-26. An active SnRK1 complex containing Gal83 as the ß-subunit and sucrose nonfermenting4 as the γ-subunit was constructed to examine functional aspects of the Adi3 interaction with SnRK1 and Gal83. These assays revealed that Adi3 is capable of suppressing the kinase activity of the SnRK1 complex through Gal83 phosphorylation plus the interaction with SnRK1 and suggested that this function may be related to the cell death suppression activity of Adi3.

SB203580, a p38 mitogen-activated protein kinase inhibitor, suppresses the development of endometriosis by down-regulating proinflammatory cytokines and proteolytic factors in a mouse model.

BACKGROUND: p38 mitogen-activated protein kinase (p38 MAPK), a regulator of inflammation, may play a role in the pathogenesis of endometriosis (EM). We studied the effect of SB203580, a p38 MAPK inhibitor, on the development of EM in a mouse model. METHODS: EM was induced in BALB/c mice by peritoneal injection of endometrium-rich fragments. Mice (n = 15) were injected i.p. for 24 days with SB203580 and 15 mice served as positive controls (EM group). Sham-operated mice received carrier only. Peritoneal fluid (PF) cells were collected for protein/mRNA analysis. Interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, matrix metalloproteinase-2 (MMP-2) and MMP-9 proteins were measured using enzyme-linked immunosorbent assay and mRNAs by RT-PCR. Phosphorylation of p38 MAPK was evaluated by western blotting. RESULTS: SB203580 decreased the weight and size (P < 0.05 versus EM) of endometriotic lesions in BALB/c mice. IL-1ß, TNF-α, MMP-2 and MMP-9 mRNA levels were decreased in peritoneal cells of the SB203580 versus EM group (P < 0.01, P < 0.05, P < 0.05 and P < 0.05, respectively). Concentrations of IL-1ß, TNF-α, MMP-2 and MMP-9 proteins in PF were reduced in the SB203580 versus EM group (P < 0.05, P < 0.01, P < 0.05 and P < 0.05, respectively). Compared with the sham-operated group, phosphorylation of p38 MAPK in the EM group was increased, and this was down-regulated by SB203580 (P < 0.01). CONCLUSIONS: SB203580 may suppress the development of EM by inhibiting expression of proinflammatory cytokines and proteolytic factors. p38 MAPK might play a key role in progression of EM.