Mutation of Residue Threonine-2 of the D2 Polypeptide and Its Effect on Photosystem II Function in Chlamydomonas reinhardtii1

The D2 polypeptide of the photosystem II (PSII) complex in the green alga Chlamydomonas reinhardtii is thought to be reversibly phosphorylated. By analogy to higher plants, the phosphorylation site is likely to be at residue threonine-2 (Thr-2). We have investigated the role of D2 phosphorylation by constructing two mutants in which residue Thr-2 has been replaced by either alanine or serine. Both mutants grew photoautotrophically at wild-type rates, and noninvasive biophysical measurements, including the decay of chlorophyll fluorescence, the peak temperature of thermoluminescence bands, and rates of oxygen evolution, indicate little perturbation to electron transfer through the PSII complex. The susceptibility of mutant PSII to photoinactivation as measured by the light-induced loss of PSII activity in whole cells in the presence of the protein-synthesis inhibitors chloramphenicol or lincomycin was similar to that of wild type. These results indicate that phosphorylation at Thr-2 is not required for PSII function or for protection from photoinactivation. In control experiments the phosphorylation of D2 in wild-type C. reinhardtii was examined by 32P labeling in vivo and in vitro. No evidence for the phosphorylation of D2 in the wild type could be obtained. [14C]Acetate-labeling experiments in the presence of an inhibitor of cytoplasmic protein synthesis also failed to identify phosphorylated (D2.1) and nonphosphorylated (D2.2) forms of D2 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results suggest that the existence of D2 phosphorylation in C. reinhardtii is still in question.

Dopamine inhibits mammalian photoreceptor Na+,K+-ATPase activity via a selective effect on the alpha3 isozyme.

The rat retina contains dopaminergic interplexiform cells that send processes to the outer plexiform layer where dopamine is released in a light-dependent manner. We report herein that physiologically relevant concentrations of dopamine inhibited ouabain-sensitive photoreceptor oxygen consumption in dark- and light-adapted rat retinas and inhibited Na+,K+-ATPase specific activity (EC 3.6.1.37) in a rat rod outer-inner segment preparation. Experiments with the selective D1 agonist fenoldopam or D2 agonist quinpirole and experiments with dopamine plus either the D1 antagonist SCH23390 or D2/D4 antagonist clozapine showed that the inhibition of oxygen consumption and enzyme activity were mediated by D2/D4-like receptors. The amphetamine-induced release of dopamine, monitored by the inhibition of oxygen consumption, was blocked by L-2-amino-4-phosphonobutyric acid and kynurenic acid. Pharmacological and biochemical experiments determined that the IC50 values of ouabain for the alpha1-low and alpha3-high ouabain affinity isozymes of photoreceptor Na+,K+-ATPase were approximately 10(-5) and approximately 10(-7) M, respectively, and that the D2/D4-like mediated inhibition of Na+,K+-ATPase was exclusively selective for the alpha3 isozyme. The dopamine-mediated inhibition of alpha3 first occurred at 5 nM, was maximal at 100 microM (-47%), had an IC50 value of 382 +/- 23 nM, and exhibited negative cooperativity (Hill coefficient, 0.27). Prior homogenization of the rod outer-inner segment completely prevented the long-lasting inhibition, suggesting that the effect was coupled to a second messenger. Although the physiological significance of our findings to photoreceptor function is unknown, we hypothesize that these results may have relevance for the temporal tuning properties of rods.

Prostaglandin H synthase 2 is expressed abnormally in human colon cancer: evidence for a transcriptional effect.

Evidence from epidemiological studies, clinical trials, and animal experiments indicates that inhibitors of prostaglandin synthesis lower the risk of colon cancer. We tested the hypothesis that abnormal expression of prostaglandin H synthase 2 (PHS-2), which can be induced by oncogenes and tumor promoters, occurs during colon carcinogenesis by examining its level in colon tumors. Human colon cancers were found to have an increased expression of PHS-2 mRNA compared with normal colon specimens from the same patient (n = 5). In situ hybridization showed that the neoplastic colonocytes had increased expression of PHS-2 (n = 4). Additionally, five colon cancer cell lines were shown to express high levels of PHS-2 mRNA even in the absence of a known inducer of PHS-2. To study the basis for this increased gene expression, we transfected a colon cancer cell line, HCT-116, with a reporter gene containing 2.0 kb of the 5' regulatory sequence of the PHS-2 gene. Constitutive transcription of the reporter gene was observed, whereas normal control cell lines transcribed the reporter only in response to an exogenous agonist. We conclude that PHS-2 is transcribed abnormally in human colon cancers and that this may be one mechanism by which prostaglandins or related compounds that support carcinogenesis are generated.