Inhibition of cyclin-dependent kinases (CDKs) by Thr14 /Tyr 15 phosphorylation is critical for normal cell cycle progression and is a converging event for several cell cycle checkpoints. In this study, we compared the relative contribution of inhibitory phosphorylation for cyclin A/B1-CDC2 and cyclin A/E-CDK2 complexes. We found that inhibitory phosphorylation plays a major role in the regulation of CDC2 but only a minor role for CDK2 during the unperturbed cell cycle of HeLa cells. The relative importance of inhibitory phosphorylation of CDC2 and CDK2 may reflect their distinct cellular functions. Despite this, expression of nonphosphorylation mutants of both CDC2 and CDK2 triggered unscheduled histone H3 phosphorylation early in the cell cycle and was cytotoxic. DNA damage by a radiomimetic drug or replication block by hydroxyurea stimulated a buildup of cyclin B1 but was accompanied by an increase of inhibitory phosphorylation of CDC2. After DNA damage and replication block, all cyclin-CDK pairs that control S phase and mitosis were to different degrees inhibited by phosphorylation. Ectopic expression of nonphosphorylated CDC2 stimulated DNA replication, histone H3 phosphorylation, and cell division even after DNA damage. Similarly, a nonphosphorylation mutant of CDK2, but not CDK4, disrupted the G 2 DNA damage checkpoint. Finally, CDC25A, CDC25B, a dominantnegative CHK1, but not CDC25C or a dominant-negative WEE1, stimulated histone H3 phosphorylation after DNA damage. These data suggest differential contributions for the various regulators of Thr 14 /Tyr 15 phosphorylation in normal cell cycle and during the DNA damage checkpoint.Cyclins and cyclin-dependent kinases (CDKs) 1 are key regulators of the eukaryotic cell cycle. In mammalian cells, different cyclin-CDK complexes are involved in regulating different cell cycle transitions: cyclin D-CDK4

BackgroundPatients with autism spectrum disorder (ASD) may have low brain serotonin concentrations as reflected by the serotonin end-metabolite 5-hydroxyindolacetic acid (5HIAA) in cerebrospinal fluid (CSF).MethodsWe sequenced the candidate genes SLC6A4 (SERT), SLC29A4 (PMAT), and GCHFR (GFRP), followed by whole exome analysis.ResultsThe known heterozygous p.Gly56Ala mutation in the SLC6A4 gene was equally found in the ASD and control populations. Using a genetic candidate gene approach, we identified, in 8 patients of a cohort of 248 with ASD, a high prevalence (3.2%) of three novel heterozygous non-synonymous mutations within the SLC29A4 plasma membrane monoamine transporter (PMAT) gene, c.86A > G (p.Asp29Gly) in two patients, c.412G > A (p.Ala138Thr) in five patients, and c.978 T > G (p.Asp326Glu) in one patient. Genome analysis of unaffected parents confirmed that these PMAT mutations were not de novo but inherited mutations. Upon analyzing over 15,000 normal control chromosomes, only SLC29A4 c.86A > G was found in 23 alleles (0.14%), while neither c.412G > A (<0.007%) nor c.978 T > G (<0.007%) were observed in all chromosomes analyzed, emphasizing the rareness of the three alterations. Expression of mutations PMAT-p.Ala138Thr and p.Asp326Glu in cellulae revealed significant reduced transport uptake activity towards a variety of substrates including serotonin, dopamine, and 1-methyl-4-phenylpyridinium (MPP+), while mutation p.Asp29Gly had reduced transport activity only towards MPP+. At least two ASD subjects with either the PMAT-Ala138Thr or the PMAT-Asp326Glu mutation with altered serotonin transport activity had, besides low 5HIAA in CSF, elevated serotonin levels in blood and platelets. Moreover, whole exome sequencing revealed additional alterations in these two ASD patients in mainly serotonin-homeostasis genes compared to their non-affected family members.ConclusionsOur findings link mutations in SLC29A4 to the ASD population although not invariably to low brain serotonin. PMAT dysfunction is speculated to raise serotonin prenatally, exerting a negative feedback inhibition through serotonin receptors on development of serotonin networks and local serotonin synthesis. Exome sequencing of serotonin homeostasis genes in two families illustrated more insight in aberrant serotonin signaling in ASD.

Depression and use of antidepressant medications are both associated with increased risk of obesity, potentially attributed to a reduced serotonin transporter (SERT) function. However, how SERT deficiency promotes obesity is unknown. Here, we demonstrated that SERT −/− mice display abnormal fat accumulation in both white and brown adipose tissues, glucose intolerance and insulin resistance while exhibiting suppressed aromatase (Cyp19a1) expression and reduced circulating 17β-estradiol levels. 17β-estradiol replacement in SERT −/− mice reversed the obesity and glucose intolerance, supporting a role for estrogen in SERT deficiency-associated obesity and glucose intolerance. Treatment of wild type mice with paroxetine, a chemical inhibitor of SERT, also resulted in Cyp19a1 suppression, decreased circulating 17β-estradiol levels, abnormal fat accumulation, and glucose intolerance. Such effects were not observed in paroxetine-treated SERT −/− mice. Conversely, pregnant SERT −/− mice displayed normalized estrogen levels, markedly reduced fat accumulation, and improved glucose tolerance, which can be eliminated by an antagonist of estrogen receptor α (ERα). Together, these findings support that estrogen suppression is involved in SERT deficiency-induced obesity and glucose intolerance, and suggest approaches to restore 17β-estradiol levels as a novel treatment option for SERT deficiency associated obesity and metabolic abnormalities.

ABSTRACT:Plasma membrane monoamine transporter (PMAT) is a polyspecific organic cation (OC) transporter that transports a variety of endogenous biogenic amines and xenobiotic cations. Previous radiotracer uptake studies showed that PMAT-mediated OC transport is sensitive to changes in membrane potential and extracellular pH, but the precise role of membrane potential and protons on PMAT-mediated OC transport is unknown. Here, we characterized the electrophysiological properties of PMAT in Xenopus laevis oocytes using a two-microelectrode voltage-clamp approach. PMAT-mediated histamine uptake is associated with inward currents under voltage-clamp conditions, and the currents increased in magnitude as the holding membrane potential became more negative. A similar effect was also observed for another cation, nicotine. Substrate-induced currents were largely independent of Na ؉ but showed strong dependence on membrane potential and pH of the perfusate. Detailed kinetic analysis of histamine uptake revealed that the energizing effect of membrane potentials on PMAT transport is mainly due to an augmentation of I max with little effect on K 0.5 . At most holding membrane potentials, I max at pH 6.0 is approximately 3-to 4-fold higher than that at pH 7.5, whereas K 0.5 is not dependent on pH. Together, these data unequivocally demonstrate PMAT as an electrogenic transporter and establish the physiological inside-negative membrane potential as a driving force for PMAT-mediated OC transport. The important role of membrane potential and pH in modulating the transport activity of PMAT toward OCs suggests that the in vivo activity of PMAT could be regulated by pathophysiological processes that alter physiological pH or membrane potential.

Plasma membrane monoamine transporter (PMAT) is a new polyspecific transporter that interacts with a wide range of structurally diverse organic cations. To map the physicochemical descriptors of cationic compounds that allow interaction with PMAT, we systematically analyzed the interactions between PMAT and three series of structural analogs of known organic cation substrates including phenylalkylamines, n-tetraalkylammonium (n-TAA) compounds, and ␤-carbolines. Our results showed that phenylalkylamines with a distance between the aromatic ring and the positively charged amine nitrogen atom of ϳ6.4 Å confer optimal interactions with PMAT, whereas studies with n-TAA compounds revealed an excellent correlation between IC 50 values and hydrophobicity. The five ␤-carbolines that we tested, which possess a pyridinium-like structure and are structurally related to the neurotoxin 1-methyl-4-phenylpyridinium, inhibited PMAT with high affinity (IC 50 values of 39.1-65.5 M). Cytotoxicity analysis further showed that cells expressing PMAT are 14-to 15-fold more sensitive to harmalan and norharmanium, suggesting that these two ␤-carbolines are also transportable substrates of PMAT. We then used computer-aided modeling to generate qualitative and quantitative three-dimensional pharmacophore models on the basis of 23 previously reported and currently identified PMAT inhibitors and noninhibitors. These models are characterized by a hydrogen bond donor and two to three hydrophobic features with distances between the hydrogen bond donor and hydrophobic features ranging between 5.20 and 7.02 Å. The consistency between the mapping results and observed PMAT affinity of a set of test compounds indicates that the models performed well in inhibitor prediction and could be useful for future virtual screening of new PMAT inhibitors.

Plasma membrane monoamine transporter (PMAT) is a polyspecific organic cation transporter in the solute carrier 29 (SLC29) family. Previous studies suggested that the major substrate recognition site is located within TM1-6 and PMAT interaction with organic cations may involve aromatic residues. In this study, we analyzed the role of tyrosine and tryptophan residues located within TM1-6 with the goal to identify potential residues involved in substrate recognition and translocation. The six Tyr and one Trp residues in this region were each mutated to alanine followed by analysis of these mutants’ membrane localization and transport activity towards 1-methyl-4-phenylpyridinium (MPP+), serotonin (5-HT) and dopamine. Two mutants, Y85A and Y112A, showed normal cell surface expression but lost their transport activity towards organic cations. At position Y85, aromatic substitution with Phe or Trp fully restored organic cation transport activity. Interestingly, at position Y112, Phe substitution is not allowed. Trp substitution at Y112 partially restored transport activity towards 5-HT and dopamine, but severely impaired MPP+ transport. Detailed kinetic analysis revealed that Trp substitution at Y85 and Y112 affected apparent binding affinity (Km) and maximal transport velocity (Vmax) in a substrate-dependent manner. Together these data suggest that Y85 and Y112 are important molecular determinants for PMAT function, and Y112 is indispensable for optimal interaction with organic cation substrates. Our analysis also suggested the involvement of transmembrane domains 1 and 2 in forming the substrate permeation pathway of PMAT.

Plasma membrane monoamine transporter (PMAT) is a polyspecific organic cation transporter belonging to the equilibrative nucleoside transporter (ENT) family. Despite its distinct substrate specificity from the classic nucleoside transporters ENT1 and 2, PMAT appears to share similar protein architecture with ENT1/2 and retains low affinity binding to classic ENT inhibitors such as nitrobenzylmercaptopurine riboside (NBMPR) and the coronary vasodilators dilazep and dipyridamole. Here we investigated the role of residue Ile89, a position known to be important for ENT interaction with dilazep, dipyridamole, and nucleoside substrates, in PMAT transport function and its interaction with classic ENT inhibitors using Madin-Darby canine kidney (MDCK) cells stably expressing human PMAT. Substitution of Ile89 in PMAT with Met, the counterpart residue in ENT1, resulted in normal plasma membrane localization and protein expression. Transport kinetic analysis revealed that I89M mutant had a 2.7-fold reduction in maximal transport velocity (Vmax) with no significant change in apparent binding affinity (Km) towards the prototype PMAT substrate 1-methyl-4-phenylpyridinium (MPP+), suggesting that I89 is an important determinant for the catalytic activity of PMAT. Dose-dependent inhibition studies further showed that the I89M mutation significantly increased PMAT’s sensitivity to dilazep by 2.5 fold without affecting its sensitivity to dipyridamole and NBMPR. Located at the extracellular end of transmembrane domain 1 of PMAT, I89 may occupy an important position close to the substrate permeation pathway and may be involved in direct interaction with the vasodilator dilazep.

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