Involvement of the Microtubule-Regulated RhoGEF GEF-H1 in the G12 family signaling pathways

Abstract

[cat] Les proteïnes G heterotrimèriques són reguladores de l'homeòstasi cel•lular. Coordinen la senyalització entre els receptors acoblats a proteïnes G (GPCR) i els seus efectors intracel.lulars. Hi ha 4 subfamflies de proteïnes G: G-alfa(i), G-alfa(q), G-alfa(s) y G-alfa(12), on cada família és capaç d'activar unes vies de senyalització diferents. Dintre de la subfamília de la G12 hi ha dos membres: G-alfa(12, i G-alfa(13), Estan implicades en diferents processos cel•lulars com poden ser la remodelació del citoesquelet d'actina i la proliferació ceJ.1ular a través de l'activació de la proteïna RhoA. RhoA és una GTPasa monomèrica que és activada per les proteïnes G12 a través de la interacció amb els factors bescanviadors de nucleòtid guanina específics de Rho (RhoGEF). La família de les Dbl-RhoGEFs és probablement la millor estudiada. Es caracteritzen per compartir un domini catalític OH i un domini PH. Dintre d'aquesta família hi ha la subfamília RH•RhoGEFs, on els seus membres poden ser estimulats directament per les proteïnes Gl2 a través del domini RH. Per altra banda, la GEF-Hl és una RhoGEF que pertany a una subfamília evolutivament propera a les RH-RhoGEFs on alguns dels seus membres poden ser estimulats per les proteïnes G. La GEF•Hl és una proteïna associada als microtúbuls on es troba en la seva forma inactiva, i l'alliberament dels microtúbuls promou un augment en la seva activitat catalítica envers a Rho. La GEF-HI participa en diferents processos cel.lulars com pot ser la regulació de la mitosi. Clàssicament les proteïnes G s'han associat a la membrana plasmàtica on desenvolupen les seves funcions, però resultats obtinguts en els últims anys han demostrat que també poden estar localitzades en altres compartiments cel•lulars. Resultats obtinguts durant el transcurs de la present tesi indiquen la proteïna G-alfa(12) es localitza en el fus mitòtic al final de la mitosi i es concentra al cos intermedi durant la citocinesi. A més a més, la pertorbació de la seva funció provoca defectes en les mitosis. També es proposa la GEF•Hl com una possible proteïna efectora de la G-alfa(12) en la mitosi ja que s'ha trobat que ambdues proteïnes colocalitzen al cos intermedi durant la citocinesi. Els resultats obtinguts han permès caracteritzar la interacció entre la proteïna G-alfa(12) i la GEF-H1 així com demostrar que la G-alfa(12) pot activar la GEF•H1.

[eng] Heterotrimeric G proteins are masters regulators of cell homeostasis. They coordinate the signaling between G protein coupled receptors (GPCRs) and their intracellular effectors. Heterotrimeric G proteins are composed of three subunits: G-alpha, G-beta, G-gamma. They function as a switches between an inactive GDP-bound state and an active GTP•bound state. Upon receptor activation, the G-alpha subunit undergoes a conformational change that leads to the exchange of GTP for GDP and the dissociation of the G-alpha subunit from the G-beta-gammadimer, allowing the subunits to activate their downstream effectors. The signal is terminated when the G-alpha subunit hydrolyzes its bound GTP to GDP and re-associates with G-beta-gamma. There are four subfamilies of G proteins: G-alpha (i), G-alpha(q), G-alpha(s) and G-alpha(12). Each subfamily can activate a different subset of effectors and signaling pathways. The subfamily of the G-alpha(12) protein is composed of two members: G-alpha(12) and G-alpha(13). They are involved in different processes such as embryonic development, cell growth, cell migration, angiogenesis processes and apoptosis. G-alpha(12) and G-alpha(13) have been linked to cellular events such as cytoskeletal rearrangements and cell proliferation through the activation of the small GTPase RhoA and moreover, they activate Rho principally through direct interaction with Rho-specific guanine nucleotide exchange factors (RhoGEFs). A growing number of RhoGEFs have recently been identified, with a majority as a members of the Dbl family of RhoGEF proteins. This proteins share in common a DH domain, the catalytic domain, and a PH domain, a pleckstrin homology domain. There are four RhoGEFs to be directly stimulated by the G-alpha(12/13) subfamily: p115RhoGEF, LARG, PDZRhoGEF and AKAP-Lbc. Three of them form the RH-RhoGEF subfamily because they share in common a RGS homology domain which is the implicated in their interaction with G12 proteins. GEF-Hl was discovered as a microtubule associated protein and belongs to the AKAP-Lbc subfamily. It has been reported to be regulated by its binding to microtubules, while microtubule-bound GEF-Hl is associated to have low activity its release from them leads to GEF-Hl activation and consequently, the activation of the small RhoGTPase RhoA. GEF-Hl can also be regulated by phosphorylation and several kinases can phosphorylate GEF-Hl in different moments of the cell. For instance, during cytokinesis GEF-Hl is phosphorylated by Aurora kinases and Cdk/Cyclin 8 in order to regulate its activity towars RhoA, due to be a high controlled spatiotemporal process. GEF-Hl is also considered a key player in the cross-talk of microtubules to actin dynamics and is reported to be involved in a variety of normal biological situations such as cell cycle regulation, and cell morphology, polarity and motility. Conventionally G proteins have been linked with the plasma membrane where they exert their functions. However, several studies during the last years have shown that Gproteins can also regulate processes in other cellular compartments. Previous results showed that G-alpha(12) was important for cell division. Indeed, the results obtained during this thesis indicate that G-alpha(12) is associated with the spindle apparatus in the late stages of mitosis and also located at the midbody during cytokinesis. Interestingly, perturbation of GUn function results in aberrant mitosis with different phenotypes such as multi nucleation, apoptosis or failed cytokinesis. On the other hand, the results obtained shown that GEF•Hl would be a possible downstream effector for GUn signaling in mitosis due to the fact that GUn interacts and activates GEF-Hl. The interaction takes place trough the DH-PH domains of GEF-Hl and GUn was able to promote the release of GEF-Hl from microtubules, where GEF•Hl is found in its inactive form, and to stimulate GEF activity of GEFHI. Furthermore, colocalization experiments showed that GU12 and GEF-Hl are located in the midbody during cytokinesis. So, we propose a mechanism for the interaction between GEF-Hl and G-alpha(12) during cytokinesis where sequentially G-alpha(12) would activate GEF-Hl in first place, and secondly G-alpha(12) would activate LARG, which is needed to complete the absicission process and is also stimulated by GUn.