Protein kinase C regulates amino acid transporter ATB0,+

Samluk, Łukasz; Czeredys, Magdalena; Skowronek, Krzysztof; Nałęcz, Katarzyna A.

doi:10.1016/j.bbrc.2012.04.106

Cited by 21 publications

(29 citation statements)

References 24 publications

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“…Interestingly, clone PSER-7F12 reacted with the same proteins of M r of 130,000 and 46,000, as recognized by PSR-45. It should be emphasized that clone 4A9 was previously shown by our group to detect an increased phosphorylation of a low affinity carnitine transporter ATB 0,+ , both in astrocytes and HEK293 cells treated with PMA [33], [36]. It has to be added that neither a clone requiring proline or lysine C-terminal to phosphoserine (PSER-16B4) nor anti-phosphothreonine antibody (PTHR-4D11) gave any positive results (not shown).…”

Section: Resultsmentioning

confidence: 89%

“…Polyclonal antibody against rOctn2 peptide 537–553 [35] and polyclonal antibody against ATB 0,+ [36] were raised and delivered by GenScript Corporation (Piscataway, NJ, USA). Monoclonal anti-flotillin-1 antibody was from BD Transduction Laboratories (San Jose, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

“…Transfection of HEK293 cells (ATTC®) with either p3xFLAG-CMV14 or plasmids carrying octn2 inserts was performed with Lipofectamine™ 2000, according to supplier protocol. The cells were cultured as given in [36].…”

Section: Methodsmentioning

confidence: 99%

“…The non-permeabilized and permeabilized cells were analysed with FITC-conjugated-concanavalin A (100 µg/ml) and anti-FLAG antibody (1∶1000), as described in [36].…”

Section: Methodsmentioning

confidence: 99%

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Caveolin-1 - A Novel Interacting Partner of Organic Cation/Carnitine Transporter (Octn2): Effect of Protein Kinase C on This Interaction in Rat Astrocytes

et al. 2013

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OCTN2 - the Organic Cation Transporter Novel family member 2 (SLC22A5) is known to be a xenobiotic/drug transporter. It transports as well carnitine - a compound necessary for oxidation of fatty acids and mutations of its gene cause primary carnitine deficiency. Octn2 regulation by protein kinase C (PKC) was studied in rat astrocytes - cells in which β-oxidation takes place in the brain. Activation of PKC with phorbol ester stimulated L-carnitine transport and increased cell surface presence of the transporter, although no PKC-specific phosphorylation of Octn2 could be detected. PKC activation resulted in an augmented Octn2 presence in cholesterol/sphingolipid-rich microdomains of plasma membrane (rafts) and increased co-precipitation of Octn2 with raft-proteins, caveolin-1 and flotillin-1. Deletion of potential caveolin-1 binding motifs pointed to amino acids 14–22 and 447–454 as the caveolin-1 binding sites within Octn2 sequence. A direct interaction of Octn2 with caveolin-1 in astrocytes upon PKC activation was detected by proximity ligation assay, while such an interaction was excluded in case of flotillin-1. Functioning of a multi-protein complex regulated by PKC has been postulated in rOctn2 trafficking to the cell surface, a process which could be important both under physiological conditions, when carnitine facilitates fatty acids catabolism and controls free Coenzyme A pool as well as in pathology, when transport of several drugs can induce secondary carnitine deficiency.

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Section: Resultsmentioning

confidence: 89%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…The non-permeabilized and permeabilized cells were analysed with FITC-conjugated-concanavalin A (100 µg/ml) and anti-FLAG antibody (1∶1000), as described in [36].…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Caveolin-1 - A Novel Interacting Partner of Organic Cation/Carnitine Transporter (Octn2): Effect of Protein Kinase C on This Interaction in Rat Astrocytes

et al. 2013

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“…Thus it is not surprising, that phosphorylation has been shown to play a role in the regulation of some epithelial AATs, for instance of the neutral and cationic amino acid transporter ATB 0,+ (SLC6A14) (Samluk et al . ), of the cystine and dibasic AAT rBAT‐b 0,+ AT (SLC3A1‐SLC7A9) (Mizoguchi et al . ), of the glycine and GABA transporter VIAAT (SLC32A1) (Bedet et al .…”

Section: Introductionmentioning

confidence: 99%

Anticipation of food intake induces phosphorylation switch to regulate basolateral amino acid transporter LAT4 (SLC43A2) function

Oparija

Rajendran

Poncet

et al. 2018

The Journal of Physiology

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Key points Amino acid absorption requires luminal uptake into and subsequent basolateral efflux out of epithelial cells, with the latter step being critical to regulate the intracellular concentration of the amino acids. The basolateral essential neutral amino acid uniporter LAT4 (SLC43A2) has been suggested to drive the net efflux of non‐essential and cationic amino acids via parallel amino acid antiporters by recycling some of their substrates; its deletion has been shown to cause defective postnatal growth and death in mice. Here we test the regulatory function of LAT4 phosphorylation sites by mimicking their phosphorylated and dephosphorylated states in Xenopus laevis oocytes and show that dephosphorylation of S274 and phosphorylation of S297 increase LAT4 membrane localization and function. Using new phosphorylation site‐specific antibodies, we observe changes in LAT4 phosphorylation in mouse small intestine that correspond to its upregulation at the expected feeding time. These results strongly suggest that LAT4 phosphorylation participates in the regulation of transepithelial amino acid absorption. Abstract The essential amino acid uniporters LAT4 and TAT1 are located at the basolateral side of intestinal and kidney epithelial cells and their transport function has been suggested to control the transepithelial (re)absorption of neutral and possibly also cationic amino acids. Uniporter LAT4 selectively transports the branched chain amino acids leucine, isoleucine and valine, and additionally methionine and phenylalanine. Its deletion leads to a postnatal growth failure and early death in mice. Since LAT4 has been reported to be phosphorylated in vivo, we hypothesized that phosphorylation regulates its function. Using Xenopus laevis oocytes, we tested the impact of LAT4 phosphorylation at Ser274 and Ser297 by expressing mutant constructs mimicking phosphorylated and dephosphorylated states. We then investigated the in vivo regulation of LAT4 in mouse small intestine using new phosphorylation site‐specific antibodies and a time‐restricted diet. In Xenopus oocytes, mimicking non‐phosphorylation of Ser274 led to an increase in affinity and apparent surface membrane localization of LAT4, stimulating its transport activity, while the same mutation of Ser297 decreased LAT4's apparent surface expression and transport rate. In wild‐type mice, LAT4 phosphorylation on Ser274 was uniform at the beginning of the inactive phase (ZT0). In contrast, at the beginning of the active phase (ZT12), corresponding to the anticipated feeding time, Ser274 phosphorylation was decreased and restricted to relatively large patches of cells, while Ser297 phosphorylation was increased. We conclude that phosphorylation of small intestinal LAT4 is under food‐entrained circadian control, leading presumably to an upregulation of LAT4 function at the anticipated feeding time.

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