Table 3.
The PK-A/14-3-3 motif in AANAT mediates cAMP activation of AANAT in intact cells
| Vector | Intact cell AANAT activity, pmol/mg of protein per h
|
Broken cell AANAT activity, nmol/mg of protein per h
|
ir-hAANAT
|
||||
|---|---|---|---|---|---|---|---|
| Cont | FSK | FSK/Cont | Cont | FSK | FSK/Cont | FSK/Cont | |
| hAANAT | 328 ± 38 | 3173 ± 850 | 9.6 ± 2.8 | 82 ± 23 | 200 ± 56 | 2.5 ± 0.1 | 2.8 ± 2.3 |
| hAANAT T31A | 166 ± 4 | 279 ± 26 | 1.7 ± 0.2 | 91 ± 14 | 106 ± 5 | 1.1 ± 0.1 | 1.2 ± 0.3 |
COS7 cells were transfected transiently with pcDNA3.1 (Invitrogen) containing either wild-type hAANAT or a mutant form (hAANAT T31A). Mutant hAANAT T31A was constructed from wild-type hAANAT cloned in pcDNA3.1 (Invitrogen), using the QuikChange kit (Stratagene). The AANAT activity in intact cells was estimated from the amount of melatonin in the media formed from 5-MT (12). AANAT activity in broken cells was assayed by using tryptamine (1 mM) as substrate (4). ir-hAANAT (immunoreactive-hAANAT) was immunodetected by antiserum As 3236. The intensity of the immunoreaction was quantitated after scanning the autoradiographs (ImageQuant 5.1). The data presented represent the mean (± range) of the results of two experiments, in which each treatment was done in duplicate. FSK, forskolin; Cont, control.