PER-T610A/S613A exhibits defects in protein phosphorylation and stability.

<p>(<b>A</b>) Head extracts were prepared from an equal number of <i>per⁰¹</i> flies expressing a wild type <i>per</i>13.2, PER-T610A/S613/S1187A, or PER-T610A/S613A transgene at the indicated time points. At ZT6, wild type PER (lane 2) is hyperphosphorylated and is declining in levels while both mutant forms of PER are hypo-to-moderately phosphorylated and appear stabilized (lanes 1 and 3). At ZT12, strictly hypophosphorylated wild type PER, presumably representing a new round of synthesis, is observed (lane 5) while mutant PER remains hypo-to-moderately phosphorylated and stable. By ZT18, mutant PER also appears in a hypophosphorylated form, though a small smear in mutant lanes is consistently observed. At ZT24, wild type PER (lane 11) is already becoming moderately phosphorylated while the mutant forms remain hypophosphorylated. As a control, <i>per⁰¹</i> flies without a transgene are shown in lane 13. Similar results were obtained for at least three independent experiments. (<b>B</b>) A time course comparison between wild type PER (<i>per</i>13.2) and PER-T610A/S613A. Head extracts were prepared from an equal number of flies every two hours at the indicated time points. When levels of wild type PER levels are lowest (top blot, ZT8-12), PER-T610A/S613A levels remain high; lowest level of PER-T610A/S613A expression is most consistently seen between ZT16-20. Note the smear at ZT14 and ZT16 in the <i>per</i>13.2 blot is not representative of phosphorylation but rather an artifact in those lanes. (<b>C</b>) The single PER-T613A mutant becomes hypophosphorylated earlier in the day than does the PER-T610A/S613A double mutant. Notice at ZT16, the single mutant (last lane) is almost completely hypophosphorylated (caret) while the double mutant remains moderately phosphorylated at this time point. Similar results were obtained from two different experiments.