Arabidopsis EDR1 Protein Kinase Regulates the Association of EDS1 and PAD4 to Inhibit Cell Death

ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4) are sequence-related lipase-like proteins that function as a complex to regulate defense responses in Arabidopsis by both salicylic acid–dependent and independent pathways. Here, we describe a gain-of-function mutation in PAD4 (S135F) that enhances resistance and cell death in response to infection by the powdery mildew pathogen Golovinomyces cichoracearum. The mutant PAD4 protein accumulates to wild-type levels in Arabidopsis cells, thus these phenotypes are unlikely to be due to PAD4 over accumulation. The phenotypes are similar to loss-of-function mutations in the protein kinase EDR1 (Enhanced Disease Resistance1), and previous work has shown that loss of PAD4 or EDS1 suppresses edr1-mediated phenotypes, placing these proteins downstream of EDR1. Here, we show that EDR1 directly associates with EDS1 and PAD4 and inhibits their interaction in yeast and plant cells. We propose a model whereby EDR1 negatively regulates defense responses by interfering with the heteromeric association of EDS1 and PAD4. Our data indicate that the S135F mutation likely alters an EDS1-independent function of PAD4, potentially shedding light on a yet-unknown PAD4 signaling function.

Arabidopsis ENHANCED DISEASE RESISTANCE1 Protein Kinase Regulates the Association of ENHANCED DISEASE SUSCEPTIBILITY1 and PHYTOALEXIN DEFICIENT4 to Inhibit Cell Death

ABSTRACTENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4) are sequence-related lipase-like proteins that function as a complex to regulate defense responses in Arabidopsis by both salicylic acid-dependent and independent pathways. Here we describe a gain-of-function mutation in PAD4 (S135F) that enhances resistance and cell death in response to infection by the powdery mildew pathogen Golovinomyces cichoracearum. The mutant PAD4 protein accumulates to wild-type levels in Arabidopsis cells, thus these phenotypes are unlikely to be due to PAD4 over accumulation. The phenotypes are similar to loss of function mutations in the protein kinase Enhanced Disease Resistance1 (EDR1), and previous work has shown that loss of PAD4 or EDS1 suppresses edr1-mediated phenotypes, placing these proteins downstream of EDR1. Here we show that EDR1 directly associates with EDS1 and PAD4 and inhibits their interaction in yeast and plant cells. We propose a model whereby EDR1 negatively regulates defense responses by interfering with the heteromeric association of EDS1 and PAD4. Our data indicate that the S135F mutation likely alters an EDS1-independent function of PAD4, potentially shedding light on a yet unknown PAD4 signaling function.

Expression of Kir2.1 Inward Rectifying Potassium Channels in Optic Nerve Glia: Evidence for Heteromeric Association with Kir4.1 and Kir5.1

Inward rectifying potassium (Kir) channels comprise a large family with diverse biophysical properties. A predominant feature of central nervous system (CNS) glia is their expression of Kir4.1, which as homomers are weakly rectifying channels, but form strongly rectifying channels as heteromers with Kir2.1. However, the extent of Kir2.1 expression and their association with Kir4.1 in glia throughout the CNS is unclear. We have examined this in astrocytes and oligodendrocytes of the mouse optic nerve, a typical CNS white matter tract. Western blot and immunocytochemistry demonstrates that optic nerve astrocytes and oligodendrocytes express Kir2.1 and that it co-localises with Kir4.1. Co-immunoprecipitation analysis provided further evidence that Kir2.1 associate with Kir4.1 and, moreover, Kir2.1 expression was significantly reduced in optic nerves and brains from Kir4.1 knock-out mice. In addition, optic nerve glia express Kir5.1, which may associate with Kir2.1 to form silent channels. Immunocytochemical and co-immunoprecipitation analyses indicate that Kir2.1 associate with Kir5.1 in optic nerve glia, but not in the brain. The results provide evidence that astrocytes and oligodendrocytes may express heteromeric Kir2.1/Kir4.1 and Kir2.1/Kir5.1 channels, together with homomeric Kir2.1 and Kir4.1 channels. In astrocytes, expression of multiple Kir channels is the biophysical substrate for the uptake and redistribution of K+ released during neuronal electrical activity known as ‘potassium spatial buffering’. Our findings suggest a similar potential role for the diverse Kir channels expressed by oligodendrocytes, which by way of their myelin sheaths are intimately associated with the sites of action potential propagation and axonal K+ release.

Heteromeric and Homomeric Transmembrane Domain Associations Regulate αIIbβ3 Function.

The integrin αIIbβ3 resides on the platelet surface in an equilibrium between inactive and active conformations that can be shifted in either direction by altering the distance between the stalks that anchor αIIbβ3 in the platelet membrane. Accordingly, the αIIb and β3 transmembrane (TM) domains, located near the ends of the stalks, are in proximity when αIIbβ3 is inactive and separate upon αIIbβ3 activation. Peptides corresponding to these domains undergo both homomeric and heteromeric interactions in biological membranes. Thus, it is possible that the shift between inactive and active αIIbβ3 conformations is accompanied by a shift from heteromeric to homomeric αIIb and β3 TM domain interactions. Indeed, we reported that introducing Asn, a residue known to strengthen homomeric TM helix interactions, into the β3 TM domain shifts αIIbβ3 to an active state. As a further test of this model of αIIbβ3 regulation, we studied the effects of mutations of the αIIb TM domain. First, we placed Asn at 10 consecutive positions in the αIIb TM domain, extending from residues V969 to L978, and co-expressed each mutant with WT β3 in CHO cells. Only one of the mutants, G972N, was constitutively active, binding ~ 8-fold more fibrinogen than WT αIIbβ3. Moreover, G972N was expressed in non-uniform patches on the CHO cell surface, consistent with the formation of αIIbβ3 clusters. G972 is the first residue of a GxxxG motif that is essential for dimerization of the αIIb TM domain. Using the TOXCAT assay to assess TM domain dimerization, we observed that G972N results in a 55% decrease in TOXCAT activity. This implies that the effect of G972N on the αIIbβ3 activation state was not a result of increased homo-dimerization of αIIb, but it is more likely that the mutation disrupted its heteromeric interaction with β3. To test this suggestion, we introduced mutations known to disrupt αIIb homo-dimerization (G972L, G976A, and G976L) into αIIbβ3 and measured their effect on αIIbβ3 function. Like G972N, each mutation induced constitutive αIIbβ3 activation and clustering. Lastly, we measured the effect of L980A, a mutation in the αIIb TM domain that unlike G972N, results in a 2.5-fold increase in TOXCAT activity. CHO cells expressing L980A constitutively bound ~ 6.5-fold more fibrinogen than did cells expressing WT αIIbβ3. Taken together, our results suggest a mechanism for αIIbβ3 regulation that involves both the heteromeric and homomeric association of the αIIb and β3 TM domains. Any process that destabilizes the heteromeric association of the αIIb and β3 TM domains would be expected to allow dissociation of these domains with concomitant αIIbβ3 activation. Hence, mutations that disrupt the heteromeric αIIb/β3 TM domain interface “push” αIIbβ3 toward activation. Conversely, intermolecular interactions that either require separation of the αIIb and β3 TM domains or are more favorable when they dissociate, such as homo-oligomerization of the αIIb and β3 TM domains, will “pull” the equilibrium toward the activated state.