La trayectoria en la experiencia

Amino acids play important roles in a plethora of different physiological process; therefore, their concentration need to be constantly regulated depending on the nutritional availability. This regulation is known to involve different metabolic pathways and physiological responses, where amino acid transporters play a central role. System A for example, is known to be influenced by hormones such as glucocorticoids, glucagon and insulin (Fehlmann et al., 1979; Varoqui & Erickson, 2002) via PKA and CREB pathways (Tovar et al., 2000; Hyde et al., 2007). Along the same line, the amino acid transporter LAT1 is known to be regulated in activated T cells via the AP-1 and NF-κB pathways (Hayashi et al., 2013). Finally, considering the amino acid intake at the level of the small intestine, B°AT1 has been shown to be regulated by glutamine and leptin in isolated rat everted gut sacs both at the mRNA and protein level (Ducroc et al., 2010). This finding complements a well-documented list of nutrient transporters regulated by leptin, i.e. MCT-1 (Buyse et al., 2002), PepT1 (Buyse et al., 2001), GLUT2, GLUT5 (Sakar et al., 2009) and SGLT1 (Lostao et al., 1998; Ducroc et al., 2005). The leptin response involves both the MAPK pathway as well as JAK2 (Morris & Rui, 2009) and experiments performed in X. laevis oocytes suggest a possible regulation of B°AT1 function by JAK2 (Bhavsar et al., 2011).

134  In our case, we aimed at understanding the regulation of LAT4 through phosphorylation at a specific serine 274 residue, which has been described in a large phosphoproteomic screening by Feric et al. (Feric et al., 2011). We approached this issue by mutagenizing the target serine residue in an alanine residue (S274A), which cannot be phosphorylated, or a glutamate residue (S274E), which should on the other hand chemically mimic a phosphorylated serine. Preliminary data in X. laevis oocytes clearly indicated that the different mutants possessed difference in LAT4 function independently on the days of expression (Fig. 5.9). Subsequent transduction in MDCK cells confirmed these findings, but, interestingly, the different mutant constructs revealed a remarkable difference in total protein expression (Fig. 5.10). The pattern of protein expression was also not directly correlating with LAT4 function, since the S274E displayed the highest protein expression but the lowest function in MDCK cells and X. laevis oocytes. This raised the possibility of a different degree of LAT4 surface expression in the different mutants; but, unfortunately, we encountered technical problems with the surface biotinylation (Fig. 5.12) and analysis of LAT4 distribution with laser confocal microscope scanning did not reveal a major difference between the different mutants (Fig. 5.14). This observation highlights the possibility that the serine phosphorylation is not essentially influencing LAT4 intracellular distribution but rather its function at the membrane. This may involve on one side a direct influence of the negatively charged phosphate group on the protein structure and thereby rendering the amino acid binding site less accessible, or, on the other side, the binding of an inhibitory protein to the phosphorylated serine residue. To investigate this issue, future experiments should be aimed at identifying and comparing the LAT4-interacting proteins in the different cell lines by performing for instance co-immunoprecipitation studies. In this sense, the candidate protein for the inhibitory function should be highly enriched in the co- immunoprecipitated fraction of the S274E mutant; whereas, it should be absent or present in low concentration in the fraction of the S274A mutant. To finally prove the involvement of the candidate protein, specific knock-down studies in MDCK should be performed to see if the reduced LAT4 function in the S274E mutant can be reverted.

In addition to investigating signaling pathways downstream of the phosphorylation step, the identification of the responsible kinase represents as well a very important issue. Interestingly, analysis of the sequence surrounding the predicted phosphorylated serine

135  residue (VGRRLS274) revealed a consensus sequence (VXRXXS) for the serine/threonine protein kinases D (PKDs). These kinases are downstream effectors of protein kinase C (PKC) and diacylglycerol (DAG) that mediate the actions of growth factors, hormones and neurotransmitters. Activated PKDs are known to be involved in different cellular mechanisms such as oxidative stress, cell motility, Golgi vesicle dynamics, transcription regulation and innate immunity (Fu & Rubin, 2011). To address the possible involvement of PKDs in LAT4 phosphorylation, preliminary studies should aim at clarifying the interaction between the kinases and the transporter with co-immunoprecipitation and co- immunofluorescence. In order to prove the functional implication of PKDs in LAT4 phosphorylation, a pivotal tool to establish is a phospho-specific anti LAT4 antibody, which could be used to quantify the amount of phosphorylated LAT4 in co-expression with different PKD isoforms in cell culture models. As a complement to these studies, one should also consider the use of PKD inhibitors (e.g. Gö6976) or siRNA-mediated knockdown of PKDs that would be expected to reduce the amount of phosphorylated LAT4.

Beyond the investigation of the cellular mechanisms leading to LAT4 phosphorylation, an essential point to elucidate is the physiological relevance and the implication of LAT4 regulation. In particular, experiments should be devoted to the specific identification of the physiological triggers (e.g. growth factors, hormones or other paracrine substances) and the functional consequences of LAT4 phosphorylation in the context of the whole body amino acid homestasis. Moreover, it is important to highlight, that some of the LAT4 substrates (i.e. branched-chain amino acids) represent important signaling molecules for the mTORC1-dependent pathway, which is known to play a central role in cell growth and division. Therefore, it is reasonable to hypothesize a functional cross-talk between mTORC1 and branched-chain amino acids, where LAT4 might play an important role.

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