Novel approaches to understanding Hent2 and hENT2-related proteins: From novelnuclear variants to global networks / Nuevos enfoques sobre el estudio de hENT2 y proteínas relacionadas: desde nuevas variantes nucleares a redes globales

Abstract

hENT2 is the human nucleoside transporter for which most splice variants have been described to date, in contrast to hENT1 for which no variants have been reported yet. In this dissertation, we have identified 3 novel splice variants of hENT2 which encode 2 novel nuclear isoforms of this transporter protein. As we demonstrated, these variants appear to be functional and ubiquitously expressed among cell lines and tissues, although the pattern of expression levels may vary depending on the growth conditions and the cellular needs.

The physiological role of these novel nuclear variants of hENT2 still remains unclear, since there is no evidence of the presence of the nucleoside salvage pathway inside the nucleus. Therefore, the presence of nucleoside transporters at the nuclear membrane might not have any relevant meaning. Nonetheless, based on the protein-protein interactions obtained from the screening with MYTH technique, other functional implications are coming up.

These nuclear variants of hENT2 could be involved in alternative splicing regulation, since they appeared to interact with several splicing factors. As we proved, hENT2 splicing patterns vary depending upon growth conditions, suggesting the existence of a regulation (or even a self-regulation) mechanism of the splicing phenomenon. Several proteins have been previously described as self-regulators of alternative splicing (Sureau et al. 2001; Sun et al. 2010; de Morree et al. 2012). In this dissertation we propose the hypothesis of HNP32 and HNP36 proteins, localized at the nuclear membrane, interacting with splice regulation factors and modulating their availability to bind pre-mRNA, thus determining the splice sites recognized by the spliceosome. In such a way, hENT2 itself would regulate the ratio between the wt isoform and the nuclear variants to adapt itself to the cellular situation.

According to our in vitro phosphorylation assays, hENT2 could be phosphorylated by CKII inside the cell. CKII is a kinase that appears to be unregulated because it is constitutively active. Therefore, it has been suggested that changes in the phosphorylation status of CKII substrates would depend on regulated dephosphorylation (Pinna 1990). Considering the putative interaction between hENT2 and PP1, we propose a possible regulation of hENT2 by phosphorylation, where the transporter would be phosphorylated by default and regulation would come by dephosphorylation via PP1. Actually, considering our biotinylation results where we found a double band at 54 KDa, the supposed phosphorylated protein would remain intracellular while the supposed non phosphorylated protein would be active at the plasma membrane.

Although we could not demonstrate a direct in vitro phosphorylation of hENT2 by PKC, this kinase appears to be involved in hENT2 trafficking regulation. Activation of PKC promotes hENT2 trafficking to the plasma membrane although it does not necessarily entails an increase of hENT2-related transport activity. Actually, the clear consequence of the hENT2 translocation to the plasma membrane is a significant decrease on hENT1 activity.

We propose the theory of a dual population of hENT2 proteins at the plasma membrane. One non-phosphorylated hENT2 isoform of 54 KDa could be related with transport activity, while another isoform of 45 KDa would not be active as a transporter, despite being present at the plasma membrane. Similarly, two different kinds of regulation by phosphorylation would affect hENT2 function, either regulating its trafficking to the plasma membrane via PKC, or activating its function as a nucleoside transporter via PP1. These two putative pathways of regulation would not necessarily be independent of each other.

Finally, our transcriptomic analysis of gastro-hepatic cell lines allowed us to place ENT2 within a global gene network involved in cell proliferation and survival. PKC appeared to be a possible central point of regulation of that network, coordinating a global response of the cell to the growth and environmental conditions. In addition, part of that novel network, including hENT2, could be involved in response to paclitaxel treatment. As we understand, a positive response to the therapy would not be determined by the hENT2 role as a transporter, but by the context of this network connecting several genes involved in proliferation and cell survival.

La bioasequibilidad y la acción de los fármacos derivados de nucleósidos utilizados en la terapia antitumoral dependen en gran medida de la capacidad de las células diana para internalizarlos y activarlos metabólicamente. Estos fármacos interfieren en los procesos de captación y metabolización de los nucleósidos naturales, incidiendo así en una de las rutas metabólicas probablemente mejor conservadas evolutivamente, la vía de recuperación, finamente regulada y de notable eficacia energética si se tiene en cuenta el coste asociado a la síntesis de novo de nucleósidos (Molina-Arcas et al. 2006).

Dado su carácter hidrofílico, tanto los nucleósidos como sus derivados requieren de transportadores de membrana para poder ser internalizados. En la presente tesis, hemos identificado 3 nuevas variantes de splicing del transportador de nucleósidos equilibrativo 2 (hENT2), las cuales codifican dos nuevas isoformas nucleares de la proteína. Dichas variantes parecen ser funcionales y estar ubicuamente expresadas en líneas celulares y tejidos sanos.

Se han realizado estudios de regulación por fosforilación del transportador hENT2 y hemos demostrado que hENT2 es fosforilado in vitro por la kinasa CKII. Dado que CKII se encuentra constitutivamente activa, hENT2 podría estar regulado por desfosforilación a través de PP1. Además, la activación de PKC mediante el tratamiento con PDD promueve la translocación de de hENT2 hacia la membrana plasmática en células HEK-293, a la vez que disminuye la actividad transportadora de hENT1. En base a nuestros resultados proponemos un modelo en que hENT2 podría tener una población dual en la membrana plasmática. De manera que una isoforma no fosforilada de hENT2, de unos 54 KDa, podría estar relacionada con la actividad transportadora, mientras que otra isoforma de 45 KDa no sería activa como transportador, a pesar de estar situada en la membrana plasmática.

Por otro lado, estudios de faramocegónimca nos han permitido definir una red global de genes aparentemente envueltos en proliferación celular y proliferación. ENT1, ENT2 y CNT3 forman parte de esta red génica, en la cual PKC podría tener un papel central regulador. ENT2, junto con otros genes implicados en la proliferación celular y supervivencia, correlacionan negativamente con la supervivencia celular en respuesta al tratamiento con paclitaxel en líneas celulares gastro-hepáticas.