Molecular cloning of a potato leaf cDNA encoding an aspartic protease (StAsp) and its expression after P. infestans infection.

Aspartic proteinases (EC 3.4.23) are widely distributed in the plant kingdom, and a number of cDNAs have been isolated from different plants. Here we report the isolation an expression analysis of a cDNA from Solanum tuberosum L. (cv. Pampeana) named StAsp. The StAsp cDNA clone was obtained using a reverse transcriptase-polymerase chain reaction (RT-PCR) and degenerated primers encoding to plant aspartic proteinases conserved domains. The coding region of the gene is 1494 bp long encoding 497 amino acids of a predicted 54 kDa molecular mass and with a pI of 5.5. The gene shares a high homology with an aspartic proteinase cDNA of tomato, 97% and 94% homology on the level of DNA and protein, respectively. The deduced amino acid sequence contains the conserved features of plant aspartic proteinases, including the plant specific insert. Northern blot analysis indicated that StAps transcripts are differentially accumulated in potato leaves after Phytophthora infestans infection in two potato cultivars with different degree of field resistance to this pathogen. In the resistant cultivar (Pampeana), induction was higher and more durable than in the susceptible cultivar (Bintje), suggesting that the StAsp level expression are associated with the resistance degree of potato cultivars to P. infestans. Results obtained previously about the induction of StAP proteins in stress conditions and these results suggest that potato aspartic proteinases are components of the plant defense response.

Metabotropic glutamate and dopamine receptors co-regulate AMPA receptor activity through PKA in cultured chick retinal neurones: effect on GluR4 phosphorylation and surface expression.

Glutamate receptor phosphorylation has been implicated in several forms of modulation of synaptic transmission. It has been reported that protein kinase A (PKA) can phosphorylate the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit GluR4 on Ser842, both in vitro and in vivo. Here, we studied the regulation of GluR4 phosphorylation and intracellular trafficking by PKA and by metabotropic receptors coupled to adenylyl cyclase (AC), in cultured chick retinal amacrine-like neurones, which are enriched in GluR4. The regulation of AMPA receptor activity by PKA and by metabotropic AC-coupled receptors was also investigated by measuring the [Ca2+]i response to kainate in Na(+)-free medium. Stimulation of AC with forskolin (FSK), or using the selective agonist of dopamine D1 receptors (+/-)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol (SKF38393), increased the [Ca2+]i response to kainate, GluR4 phosphorylation at Ser842 and GluR4 surface expression. Pre-incubation of the cells with (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV), an agonist of group II metabotropic glutamate receptors (mGluR), which are coupled to inhibition of AC, inhibited the effect of FSK and of SKF38393 on AMPA receptor activity, GluR4 phosphorylation and expression at the plasma membrane. These results indicate that there is a functional cross-talk between dopamine D1 receptors and group II mGluR in the regulation of GluR4 phosphorylation and AMPA receptor activity. Our data show that GluR4 phosphorylation at Ser842 by PKA, and its recruitment to the plasma membrane upon phosphorylation, is regulated by metabotropic receptors.

Phosphorylation of GluR4 AMPA-type glutamate receptor subunit by protein kinase C in cultured retina amacrine neurons.

We have previously reported that the activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors is potentiated by protein kinase C (PKC) in cultured chick retina amacrine neurons, and that constitutive PKC activity is necessary for basal AMPA receptor activity (Carvalho et al., 1998). In this study, we evaluated the phosphorylation of the GluR4 subunit, which is very abundant in cultured amacrine neurons, to correlate it with the effects of PKC on AMPA receptor activity in these cells. 32P-labelling of GluR4 increased upon AMPA receptor stimulation or cell treatment with phorbol 12-myristate 13-acetate (PMA) before stimulating with kainate. By contrast, phosphorylation of GluR4 was not changed when PKC was inhibited by treating the cells with the selective PKC inhibitor GF 109203X before stimulation with kainate. We conclude that GluR4 is phosphorylated upon PKC activation and/or stimulation of AMPA receptors in cultured amacrine cells. Additionally, AMPA receptor activation with kainate in cultured chick amacrine cells leads to translocation of conventional and novel PKC isoforms to the cell membrane, suggesting that PKC could be activated upon AMPA receptor stimulation in these cells.