Cellular Stability of Serotonin N-Acetyltransferase Conferred by Phosphonodifluoromethylene Alanine (Pfa) Substitution for Ser-205

Abstract

Large changes in the activity of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) in the pineal gland control the rhythmic production of the timekeeping hormone melatonin. The activity of AANAT reflects changes in the amount and activation state of the AANAT protein, both of which increase at night. The molecular basis of this regulation is now becoming known, and recent data indicate that this involves phosphorylation-dependent binding to the 14-3-3 protein at two sites, one of which, Ser-205, is located several residues from the C terminus. In this study, we determined whether substitution of this residue with a non-hydrolyzable the phosphoserine/ phosphothreonine mimetic would promote binding to the 14-3-3 protein and enhance cellular stability. To accomplish this, a C-terminal AANAT peptide containing the phosphonodifluoromethylene alanine at Ser-205 was synthesized and fused to bacterially expressed AANAT 30 -199 using expressed protein ligation. The resulting semisynthetic protein has enhanced affinity for the expressed 14-3-3 protein and exhibits greater cellular stability in microinjection experiments, as compared with the unmodified AANAT. Enhanced 14-3-3 binding was also observed using humanized ovine AANAT, which has a different C-terminal sequence (Gly-Cys) than the ovine enzyme (Asp-Arg), indicating that that characteristic is not unique to the ovine enzyme. These studies provide the first evidence that substitution of Ser-205 with the stable phosphomimetic amino acid phosphonodifluoromethylene alanine enhances binding to 14-3-3 and the cellular stability of AANAT and are consistent with the view that Ser-205 phosphorylation plays a critical role in the regulation of AANAT activity and melatonin production.

Photic Regulation of ArylalkylamineN-Acetyltransferase Binding to 14-3-3 Proteins in Retinal Photoreceptor Cells

“…8). This is consistent with previous findings that 14-3-3 proteins protect AANAT from degradation (Zheng et al, 2003(Zheng et al, , 2005. Similarly, pT 29 -AANAT immunoreactivity was preserved during the incubation in control samples but completely disappeared in the samples incubated with R18.…”

“…These two sequences correspond to those in ovine AANAT that mediate binding to 14-3-3 (Ganguly et al, , 2005Zheng et al, 2003Zheng et al, , 2005.…”

“…Roseboom et al (1994) showed that 14-3-3 copurified with AANAT from sheep pineal glands. Subsequent studies demonstrated that phosphorylation-dependent binding to 14-3-3 proteins activates AANAT and protects it from proteolytic degradation (Ganguly et al, , 2005Zheng et al, 2003Zheng et al, , 2005. In this study, we investigated the role of 14-3-3 complex formation in the photic regulation of AANAT in chicken photoreceptor cells.…”

Efficient ADAM22 surface expression is mediated by phosphorylation-dependent interaction with 14-3-3 protein family members

“…14-3-3 protein interactions can alter ADAM22 stability In several cases 14-3-3 binding has been shown to increase stability of target proteins (Jeanclos et al, 2001;Wang et al, 2004;Zheng et al, 2005). HEK 293T cells transiently expressing either FLAG-tagged ADAM22 wild-type or the double 14-3-3 binding site mutant were treated with puromycin for up to 8 hours to inhibit protein synthesis.…”

Semisynthesis of unnatural amino acid mutants of paxillin: Protein probes for cell migration studies

“…There are several examples in which NCL has been applied to create probes for the study of protein phosphorylation. These include the semisynthesis of phosphatase-resistant phosphoprotein analogs (Lu et al 2003;Zheng et al 2005) and a dually caged phosphoSer-containing derivative of a domain of the protein Smad2 (Hahn and Muir 2004). With the semisynthesis of paxillin analogs, we demonstrate that NCL can also be applied for the assembly of caged and phosphorylated variants of full-length proteins, exemplified by targeting a large, multidomain adaptor protein.…”