Biomedical subjects

Tatiana Sorkina

Publications and source records attributed to Tatiana Sorkina.

8 recordsLinked to original sources

PubMed2006

Three ubiquitin conjugation sites in the amino terminus of the dopamine transporter mediate protein kinase C-dependent endocytosis of the transporter.

Dopamine levels in the brain are controlled by the plasma membrane dopamine transporter (DAT). The amount of DAT at the cell surface is determined by the relative rates of its internalization and recycling. Activation of protein kinase C (PKC) leads to acceleration of DAT endocytosis. We have recently demonstrated that PKC activation also results in ubiquitylation of DAT. To directly address the role of DAT ubiquitylation, lysine residues in DAT were mutated. Mutations of each lysine individually did not affect ubiquitylation and endocytosis of DAT. By contrast, ubiquitylation of mutants carrying multiple lysine substitutions was reduced in cells treated with phorbol ester to the levels detected in nonstimulated cells. Altogether, mutagenesis data suggested that Lys19, Lys27, and Lys35 clustered in the DAT amino-terminus are the major ubiquitin-conjugation sites. The data are consistent with the model whereby at any given time only one of the lysines in DAT is conjugated with a short ubiquitin chain. Importantly, cell surface biotinylation, immunofluorescence and down-regulation experiments revealed that PKC-dependent internalization of multilysine mutants was essentially abolished. These data provide the first evidence that the ubiquitin moieties conjugated to DAT may serve as a molecular interface of the transporter interaction with the endocytic machinery.

Amino Acid Substitution↗

PubMed2006

RNA interference screen reveals an essential role of Nedd4-2 in dopamine transporter ubiquitination and endocytosis.

The function of the dopamine transporter (DAT) to terminate dopamine neurotransmission is regulated by endocytic trafficking of DAT. To elucidate the mechanisms of DAT endocytosis, we generated a fully functional mutant of the human DAT in which a hemagglutinin epitope (HA) was incorporated into the second extracellular loop. The endocytosis assay, based on the uptake of an HA antibody, was designed to study constitutive- and protein kinase C (PKC)-dependent internalization of HA-DAT expressed in non-neuronal cells and rat dopaminergic neurons. Large-scale RNA interference analysis of PKC-dependent endocytosis of HA-DAT revealed the essential and specific role of an E3 ubiquitin ligase, Nedd4-2 (neural precursor cell expressed, developmentally downregulated 4-2), as well as the involvement of adaptor proteins present in clathrin-coated pits, such as epsin, Eps15 (epidermal growth factor pathway substrate clone 15), and Eps15R (Eps15-related protein). Depletion of Nedd4-2 resulted in a dramatic reduction of PKC-dependent ubiquitination of DAT. Endogenous Nedd4-2, epsin, and Eps15 were coimmunoprecipitated with heterologously expressed human HA-DAT and endogenous DAT isolated from rat striatum. A new mechanistic model of DAT endocytosis is proposed whereby the PKC-induced ubiquitination of DAT mediated by Nedd4-2 leads to interaction of DAT with adaptor proteins in coated pits and acceleration of DAT endocytosis.

Animals↗

PubMed2005

Enhanced ubiquitylation and accelerated degradation of the dopamine transporter mediated by protein kinase C.

Dopamine transporter (DAT) localization in dopaminergic neurons plays an important role in regulating dopamine signaling. However, the mechanisms of DAT trafficking that control DAT localization are still poorly understood. To gain insight into these mechanisms, human DAT was purified in large amounts using a two-step affinity chromatography procedure from untreated HeLa cells or cells treated with phorbol 12-myristate 13-acetate (PMA). Mass spectrometric analysis of purified DAT complexes revealed the presence of several proteins, among which ubiquitin was particularly abundant in the PMA-treated sample. Western blotting of highly purified DAT protein confirmed constitutive ubiquitylation of DAT and a dramatic increase in DAT ubiquitylation in cells treated with PMA. This increase was blocked by pretreatment with the protein kinase C (PKC) inhibitor bis-indolylmaleimide. DAT ubiquitylation by ectopically expressed ubiquitin was demonstrated in cells transiently transfected with yellow fluorescent protein-tagged ubiquitin. In addition, fluorescence resonance energy transfer was detected between cyan fluorescent protein-tagged DAT and yellow fluorescent protein-tagged ubiquitin, indicative of DAT-ubiquitin conjugation. Interestingly, the largest fluorescence resonance energy transfer signals were observed in endosomes. Ubiquitylated DAT was detected in the plasma membrane using cell surface biotinylation as well as in intracellular compartments, suggesting that ubiquitylation begins at the plasma membrane and is maintained in endosomes. In both porcine aortic endothelial and HeLa cells, where PKC-dependent DAT ubiquitylation was observed, PKC activation resulted in rapid degradation of DAT (t12 = 1-2 h). Altogether, these data suggest that PKC-induced DAT ubiquitylation may target DAT to lysosomal degradation.

Aggrecans↗

PubMed2005

Constitutive and protein kinase C-induced internalization of the dopamine transporter is mediated by a clathrin-dependent mechanism.

The amount of dopamine transporter (DAT) present at the cell surface is rapidly regulated by the rates of DAT internalization to endosomes and DAT recycling back to the plasma membrane. The re-distribution of the transporter from the cell surface to endosomes was induced by phorbol ester activation of protein kinase C in porcine aortic endothelial cells stably expressing the human DAT. Inhibition of DAT recycling with the carboxylic ionophore monensin also caused significant accumulation of DAT in early endosomes and a concomitant loss of DAT from the cell surface, due to protein kinase C-independent constitutive internalization of DAT in the absence of recycling. Such monensin-induced relocation of DAT to endosomes was therefore utilized as a measure of the constitutive internalization of DAT. Knock-down of clathrin heavy chain or dynamin II by small interfering RNAs dramatically inhibited both constitutive and protein kinase C-mediated internalization of DAT. In contrast, neither monensin-dependent nor phorbol ester-induced re-distribution of DAT were affected by inhibitors of endocytosis through cholesterol-rich membrane microdomains. Mutational analysis revealed the potential importance of amino acid residues 587-597 in DAT internalization. Altogether, the data suggest that both constitutive and protein kinase C-mediated internalization of DAT utilize the clathrin-dependent endocytic pathway, but likely involve unconventional mechanisms.

Amino Acid Sequence↗

PubMed2004

Multiple molecular determinants in the carboxyl terminus regulate dopamine transporter export from endoplasmic reticulum.

The plasma membrane dopamine transporter (DAT) has an essential role in terminating dopaminergic neurotransmission by reuptake of dopamine into the presynaptic neurons. Therefore, the amount of DAT at the cell surface is a critical determinant of DAT function. In this study, we examined the role of the carboxyl terminus of DAT in trafficking of the transporter through the biosynthetic pathway to the plasma membrane. Live cell fluorescence microscopy and cell surface biotinylation were used to study the effects of systematic deletions and alanine substitutions in the carboxyl terminus on DAT localization. It was found that alanine substitutions of Lys-590 and Asp-600 significantly delayed the delivery of DAT to the plasma membrane because of retention of DAT in the endoplasmic reticulum (ER). Most surprising, mutation of Gly-585 to alanine completely blocked the exit of DAT from the ER and surface expression of the transporter. The effect of these three mutations on ER export of DAT was demonstrated in porcine aortic endothelial cells and the immortalized neuronal cell line 1RB3AN27. In primary cultures of rat embryonic midbrain neurons, DAT G585A, K590A, and D600A mutants were restricted to the cell soma and did not traffic to the dendrites or axonal processes. These data are consistent with the model whereby the local conformation and/or intramolecular interactions of the sequences of the DAT carboxyl terminus proximal to the last transmembrane domain are essential for the ER export of the transporter.

Alanine↗

PubMed2003

Oligomerization of dopamine transporters visualized in living cells by fluorescence resonance energy transfer microscopy.

To examine the oligomeric state and trafficking of the dopamine transporter (DAT) in different compartments of living cells, human DAT was fused to yellow (YFP) or cyan fluorescent protein (CFP). YFP-DAT and CFP-DAT were transiently and stably expressed in porcine aortic endothelial (PAE) cells, human embryonic kidney (HEK) 293 cells, and an immortalized dopaminergic cell line 1RB3AN27. Fluorescence microscopic imaging of cells co-expressing YFP-DAT and CFP-DAT revealed fluorescence resonance energy transfer (FRET) between CFP and YFP, which is consistent with an intermolecular interaction of DAT fusion proteins. FRET signals were detected between CFP- and YFP-DAT located at the plasma membrane and in intracellular membrane compartments. Phorbol esters or amphetamine induced the endocytosis of YFP/CFP-DAT to early and recycling endosomes, identified by Rab5, Rab11, Hrs and EEA.1 proteins. Interestingly, however, DAT was mainly excluded from Rab5- and Hrs-containing microdomains within the endosomes. The strongest FRET signals were measured in endosomes, indicative of efficient oligomerization of internalized DAT. The intermolecular DAT interactions were confirmed by co-immunoprecipitation. A DAT mutant that was retained in the endoplasmic reticulum (ER) after biosynthesis was used to show that DAT is oligomeric in the ER. Moreover, co-expression of an ER-retained DAT mutant and wild-type DAT resulted in the retention of wild-type DAT in the ER. These data suggest that DAT oligomers are formed in the ER and then are constitutively maintained both at the cell surface and during trafficking between the plasma membrane and endosomes.

Amphetamines↗

PubMed2002

Effect of tyrosine kinase inhibitors on clathrin-coated pit recruitment and internalization of epidermal growth factor receptor.

Several inhibitors of epidermal growth factor receptor (EGFR) kinase and Src family kinases (SFK) were employed to study the role of these kinases in EGFR internalization through clathrin-coated pits. The EGFR kinase-specific compound PD158780 substantially diminished EGFR internalization. PP2, an inhibitor of SFK, had a moderate effect on EGFR internalization in several types of cells, including cells lacking SFK, indicating that the inhibition of endocytosis by PP2 is mediated by kinases other than SFK. In contrast, SU6656, a more specific inhibitor of SFK, did not affect EGFR internalization. To examine what stage of internalization requires receptor kinase activity, we established a quantitative assay based on three-dimensional fluorescence microscopy that measures co-localization of an EGF-rhodamine conjugate and a fluorescently tagged clathrin adaptor protein complex, AP-2. Interestingly, recruitment of EGFR into coated pits did not require physiological temperature because the maximal accumulation of EGFR in coated pits was observed at 4 degrees C. Pretreatment of the cells with PD158780 prevented EGFR recruitment into coated pits, whereas the inhibitor did not block the internalization of receptors that had first been allowed to enter the coated pits at 4 degrees C. These data demonstrate that the activation of receptor kinase is essential for the initial, coated pit recruitment step of endocytosis.

3T3 Cells↗

PubMed2002

Calmodulin regulates intracellular trafficking of epidermal growth factor receptor and the MAPK signaling pathway.

The epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase receptor family involved in signal transduction and the regulation of cellular proliferation and differentiation. It is also a calmodulin-binding protein. To examine the role of calmodulin in the regulation of EGFR, the effect of calmodulin antagonist, W-13, on the intracellular trafficking of EGFR and the MAPK signaling pathway was analyzed. W-13 did not alter the internalization of EGFR but inhibited its recycling and degradation, thus causing the accumulation of EGF and EGFR in enlarged early endosomal structures. In addition, we demonstrated that W-13 stimulated the tyrosine phosphorylation of EGFR and consequent recruitment of Shc adaptor protein with EGFR, presumably through inhibition of the calmodulin-dependent protein kinase II (CaM kinase II). W-13-mediated EGFR phosphorylation was blocked by metalloprotease inhibitor, BB94, indicating a possible involvement of shedding in this process. However, MAPK activity was decreased by W-13; dissection of this signaling pathway showed that W-13 specifically interferes with Raf-1 activity. These data are consistent with the regulation of EGFR by calmodulin at several steps of the receptor signaling and trafficking pathways.

Animals↗