Signalling by neurotrophins and hepatocyte growth factor regulates axon morphogenesis by differential β-catenin phosphorylation

Tyrosine phosphorylation of ß-catenin, a component of adhesion complexes and the Wnt pathway, affects cell adhesion, migration and gene transcription. By reducing ßcatenin availability using shRNA-mediated gene silencing or expression of intracellular N-cadherin, we show that ß-catenin is required for axon growth downstream of Brain Derived Neurotrophic Factor (BDNF) and Hepatocyte Growth Factor (HGF) signalling. We demonstrate that receptor tyrosine kinases (RTK) Trk and Met interact with and phosphorylate ß-catenin. Neurotrophins (NT) stimulation of Trk receptors results in phosphorylation of ß-catenin at residue Y654 and increased axon growth and branching. Conversely, pharmacological inhibition of Trk or a Y654F mutant blocks these effects. ß-catenin phospho(P)-Y654 colocalizes with the cytoskeleton at growth cones. However, HGF that also increases axon growth and branching, induces ß-catenin phosphorylation at Y142 and a nuclear localization. Interestingly, dominant negative ΔN-TCF4 abolishes the effects of HGF in axon growth and branching, but not of NT. We conclude that NT and HGF signalling differentially phosphorylate ß-catenin, targeting ß-catenin to distinct compartments to regulate axon morphogenesis by TCF4-transcription-dependent and independent mechanisms. These results place ß-catenin downstream of growth factor/RTK signalling in axon differentiation.

Oncogenic K-Ras segregates at spatially distinct plasma membrane signaling platforms according to its phosphorylation status

Activating mutations in the K-Ras small GTPase are extensively found in human tumors. Although these mutations induce the generation of a constitutively GTP-loaded, active form of K-Ras, phosphorylation at Ser181 within the C-terminal hypervariable region can modulate oncogenic K-Ras function without affecting the in vitro affinity for its effector Raf-1. In striking contrast, K-Ras phosphorylated at Ser181 shows increased interaction in cells with the active form of Raf-1 and with p110α, the catalytic subunit of PI 3-kinase. Because the majority of phosphorylated K-Ras is located at the plasma membrane, different localization within this membrane according to the phosphorylation status was explored. Density-gradient fractionation of the plasma membrane in the absence of detergents showed segregation of K-Ras mutants that carry a phosphomimetic or unphosphorylatable serine residue (S181D or S181A, respectively). Moreover, statistical analysis of immunoelectron microscopy showed that both phosphorylation mutants form distinct nanoclusters that do not overlap. Finally, induction of oncogenic K-Ras phosphorylation - by activation of protein kinase C (PKC) - increased its co-clustering with the phosphomimetic K-Ras mutant, whereas (when PKC is inhibited) non-phosphorylated oncogenic K-Ras clusters with the non-phosphorylatable K-Ras mutant. Most interestingly, PI 3-kinase (p110α) was found in phosphorylated K-Ras nanoclusters but not in non-phosphorylated K-Ras nanoclusters. In conclusion, our data provide - for the first time - evidence that PKC-dependent phosphorylation of oncogenic K-Ras induced its segregation in spatially distinct nanoclusters at the plasma membrane that, in turn, favor activation of Raf-1 and PI 3-kinase.

Phosphorylation of unique domains of Src family of kinases

Members of the Src family of kinases (SFKs) are non-receptor tyrosine kinases involved in numerous signal transduction pathways. The catalytic, SH3 and SH2 domains are attached to the membrane-anchoring SH4 domain through the intrinsically disordered"Unique" domains, which exhibit strong sequence divergence among SFK members. In the last decade, structural and biochemical studies have begun to uncover the crucial role of the Unique domain in the regulation of SFK activity. This mini-review discusses what is known about the phosphorylation events taking place on the SFK Unique domains, and their biological relevance. The modulation by phosphorylation of biologically relevant inter- and intra- molecular interactions of Src, as well as the existence of complex phosphorylation/dephosphorylation patterns observed for the Unique domain of Src, reinforces the important functional role of the Unique domain in the regulation mechanisms of the Src kinases and, in a wider context, of intrinsically disordered regions in cellular processes.

SMRT-mediated co-shuttling enables export of class IIa HDACs independent of their CaM kinase phosphorylation sites

The Class IIa histone deacetylases (HDAC)4 and HDAC5 play a role in neuronal survival and behavioral adaptation in the CNS. Phosphorylation at 2/3 N-terminal sites promote their nuclear export. We investigated whether non-canonical signaling routes to Class IIa HDAC export exist because of their association with the co-repressor Silencing Mediator Of Retinoic And Thyroid Hormone Receptors (SMRT). We found that, while HDAC5 and HDAC4 mutants lacking their N-terminal phosphorylation sites (HDAC4(MUT), HDAC5(MUT)) are constitutively nuclear, co-expression with SMRT renders them exportable by signals that trigger SMRT export, such as synaptic activity, HDAC inhibition, and Brain Derived Neurotrophic Factor (BDNF) signaling. We found that SMRT's repression domain 3 (RD3) is critical for co-shuttling of HDAC5(MUT), consistent with the role for this domain in Class IIa HDAC association. In the context of BDNF signaling, we found that HDAC5(WT), which was more cytoplasmic than HDAC5(MUT), accumulated in the nucleus after BDNF treatment. However, co-expression of SMRT blocked BDNF-induced HDAC5(WT) import in a RD3-dependent manner. In effect, SMRT-mediated HDAC5(WT) export was opposing the BDNF-induced HDAC5 nuclear accumulation observed in SMRT's absence. Thus, SMRT's presence may render Class IIa HDACs exportable by a wider range of signals than those which simply

Functional EF-hands in neuronal calcium sensor GCAP2 determine its phosphorylation state and subcellular distribution in vivo, and are essential for photoreceptor cell integrity

The neuronal calcium sensor proteins GCAPs (guanylate cyclase activating proteins) switch between Ca2+-free and Ca2+-bound conformational states and confer calcium sensitivity to guanylate cyclase at retinal photoreceptor cells. They play a fundamental role in light adaptation by coupling the rate of cGMP synthesis to the intracellular concentration of calcium. Mutations in GCAPs lead to blindness. The importance of functional EF-hands in GCAP1 for photoreceptor cell integrity has been well established. Mutations in GCAP1 that diminish its Ca2+ binding affinity lead to cell damage by causing unabated cGMP synthesis and accumulation of toxic levels of free cGMP and Ca2+. We here investigate the relevance of GCAP2 functional EF-hands for photoreceptor cell integrity. By characterizing transgenic mice expressing a mutant form of GCAP2 with all EF-hands inactivated (EF(-)GCAP2), we show that GCAP2 locked in its Ca2+-free conformation leads to a rapid retinal degeneration that is not due to unabated cGMP synthesis. We unveil that when locked in its Ca2+-free conformation in vivo, GCAP2 is phosphorylated at Ser201 and results in phospho-dependent binding to the chaperone 14-3-3 and retention at the inner segment and proximal cell compartments. Accumulation of phosphorylated EF(-)GCAP2 at the inner segment results in severe toxicity. We show that in wildtype mice under physiological conditions, 50% of GCAP2 is phosphorylated correlating with the 50% of the protein being retained at the inner segment. Raising mice under constant light exposure, however, drastically increases the retention of GCAP2 in its Ca2+-free form at the inner segment. This study identifies a new mechanism governing GCAP2 subcellular distribution in vivo, closely related to disease. It also identifies a pathway by which a sustained reduction in intracellular free Ca2+ could result in photoreceptor damage, relevant for light damage and for those genetic disorders resulting in 'equivalent-light'' scenarios.

Phosphorylation at Ser-181 of oncogenic KRAS is required for tumor growth

KRAS phosphorylation has been reported recently to modulate the activity of mutant KRAS protein in vitro. In this study, we defined S181 as a specific phosphorylation site required to license the oncogenic function of mutant KRAS in vivo. The phosphomutant S181A failed to induce tumors in mice, whereas the phosphomimetic mutant S181D exhibited an enhanced tumor formation capacity, compared with the wild-type KRAS protein. Reduced growth of tumors composed of cells expressing the nonphosphorylatable KRAS S181A mutant was correlated with increased apoptosis. Conversely, increased growth of tumors composed of cells expressing the phosphomimetic KRAS S181D mutant was correlated with increased activation of AKT and ERK, two major downstream effectors of KRAS. Pharmacologic treatment with PKC inhibitors impaired tumor growth associated with reduced levels of phosphorylated KRAS and reduced effector activation. In a panel of human tumor cell lines expressing various KRAS isoforms, we showed that KRAS phosphorylation was essential for survival and tumorigenic activity. Furthermore, we identified phosphorylated KRAS in a panel of primary human pancreatic tumors. Taken together, our findings establish that KRAS requires S181 phosphorylation to manifest its oncogenic properties, implying that its inhibition represents a relevant target to attack KRAS-driven tumors.

Regulació del factor de transcripció NFAT5 per les quinases WNK1, SPAK i OSR1 i cerca de quinases activadores de la pròpia WNK1 en resposta a estrés osmòtic

Estudi elaborat a partir d’una estada a la School of Life Sciences de la University of Dundee, Gran Bretanya, entre gener i març del 2007.L'estrès osmòtic causa rà pidament l'activació de la quinasa WNK1, que fosforila i activa a continuació les quinases SPAK i OSR1, que alhora regulen canals i transportadors d’ions preexistents a la membrana cel•lular. El factor de transcripció NFAT5 és el principal regulador de la resposta cel•lular transcripcional secundà ria a hipertonicitat i s’ha descrit que les quinases p38, Fyn, PKA, ERK/MEK i ATM estan involucrades en la seva regulació post-traduccional. No obstant, com que la funció d’aquestes quinases no explica totalment els mecanismes d'activació de NFAT5, s’ha estudiat si l’activitat transcripcional de NFAT5 pot estar regulada per WNK1, SPAK o OSR1. Així doncs, es va observar que l’activitat d’un reporter dependent de NFAT5 no es veu afectada per la presència de cap de les quinases anteriors, en la seva forma wild-type o dominant negatiu. D’altra banda, es va estudiar quin domini de WNK1 és necessari per a que pugui respondre a hipertonicitat i quines quinases poden estar involucrades en la fosforilació de la serina 382 de WNK1. En conclusió, les dades obtingudes apunten que l’activació de WNK1 en resposta a estrès osmòtic requereix la seva fosforilació en la serina 382 per quinases upstream com PAK2 o RSK i que també és necessari un dels seus dominis coiled-coil, almenys els aminoà cids 558 i 561. Aquests processos, però, semblen ser independents de l’activació de NFAT5 en resposta a hipertonicitat.   

Akt fosforila FoxG1 en cèl·lules de glioma

En un treball recent s’ha descrit l’amplificació del gen del factor de transcripció FoxG1, homòleg de l'oncogen víric aviar Qin, en mostres de meduloblastoma, un tipus de tumor cerebral que representa el 20% dels tumors cerebrals infantils malignes (Adesina et al.¸2007). El tumor cerebral més freqüent i agressiu en l’adult és el glioma, especialment la seva forma més maligna: el glioblastoma multiforme (glioma de grau IV segons la classificació de l'OMS). En aquest treball hem estudiat l'expressió proteica del factor de transcripció FoxG1, homòleg de l'oncogen víric aviar Qin, en mostres de glioma. Vam analitzar 15 mostres de glioma, detectant FoxG1 en 9 d’elles, i amb diferents nivells d’expressió. Intentant aprofundir en el coneixement de la funció i la regulació de FoxG1, vam estudiar si FoxG1 podia ser fosforilat. Vam detectar, tant per assaig cinasa com per espectrometria de masses, que FoxG1 és un substracte directe de la cinasa Akt, el principal efector de la via de PI3K (phosphoinositide 3-kinase). En la línia cel•lular de glioblastoma U373MG, vam observar que Akt endogen fosforila FoxG1 en un pèptid situat a l’extrem C-terminal del domini forkhead. Aquesta fosforilació és contrarestada per un inhibidor farmacològic de PI3K. Al contrari del que passa en FoxO on la fosforilació per Akt inhibeix l’activitat de FoxO promovent la seva exportació del nucli, la fosforilació de FoxG1 per Akt no promou cap canvi en la seva localització subcel•lular, i FoxG1 es manté nuclear. Actualment estem estudiant els efectes biològics de la fosforilació de FoxG1 per Akt.

Estudi de les alteracions en la capacitat neuroprotectora dels astròcits reactius en l'envelliment

En el periodo 2005-2008 hemos publicado tres artículos sobre las alteraciones de los astrocitos reactivos en el cerebro durante el envejecimiento. En el primer estudio, evaluamos la capacidad neuroprotectora de los astrocitos en un modelo experimental in vitro de envejecimiento. Los cambios en el estrés oxidativo, la captación del glutamato y la expresión proteica fueron evaluados en los astrocitos corticales de rata cultivados durante 10 y 90 días in vitro (DIV). Los astrocitos envejecidos tenían una capacidad reducida de mantener la supervivencia neuronal. Estos resultados indican que los astrocitos pueden perder parcialmente su capacidad neuroprotectora durante el envejecimiento. En el segundo estudio el factor neurotrófico derivado de la línea glial (GDNF) fue probado para observar sus efectos neurotróficos contra la atrofia neuronal que causa déficits cognitivos en la vejez. Las ratas envejecidas Fisher 344 con deficiencias en el laberinto de Morris recibieron inyecciones intrahippocampales de un vector lentiviral que codifica GDNF humano en los astrocitos o del mismo vector que codifica la proteína fluorescente verde humana como control. El GDNF secretado por los astrocitos mejoró la función de la neurona como se muestra por aumentos locales en la síntesis de los neurotransmisores acetilcolina, dopamina y serotonina. El aprendizaje espacial y la prueba de memoria demostraron un aumento significativo en las capacidades cognitivas debido a la exposición de GDNF, mientras que las ratas control mantuvieron sus resultados al nivel del azar. Estos resultados confirman el amplio espectro de la acción neurotrófica del GDNF y abre nuevas posibilidades de terapia génica para reducir la neurodegeneración asociada al envejecimiento. En el último estudio, examinamos cambios en la fosforilación de tau, el estrés oxidativo y la captación de glutamato en los cultivos primarios de astrocitos corticales de ratones neonatos de senescencia acelerada (SAMP8) y ratones resistentes a la senescencia (SAMR1). Nuestros resultados indican que las alteraciones en cultivos del astrocitos de los ratones SAMP8 son similares a las detectadas en cerebros enteros de los ratones SAMP8 de 1-5 meses de edad. Por otra parte, nuestros resultados sugieren que esta preparación in vitro es adecuada para estudiar en este modelo murino el envejecimiento temprano y sus procesos moleculares y celulares.

Funcions de les molècules associades a la mielina i la proteïna priònica cel•lular (PrPc) en neurodegeneració (Malaltia d’Alzheimer) i neuroinflamació (Esclerosi Múltiple)

Les proteïnes associades a la mielina (MAIS), Nogo-A, MAG i OMgp, són molècules que presenten una capacitat inhibitòria molt important per el recreixement axonal i la neuroreparació després de lesió. No obstant des de fa anys les seves funcions han estat ampliades i s’han involucrat en diferents processos degeneratius del sistema nerviós o en processos neuroinflamatoris del sistema nerviós central i el perifèric com ara l'Escleresi Múltiple (MS). La base neurobiològica d’indicadors moleculars que són responsables del dany axonal en MS segueixen sense estar plenament descrits. Recentment s’ha publicat que el mecanisme de senyalització Nogo-A pot regir els primers canvis de la desmielinització immunomediada del sistema nerviós central en el model animal de MS, l’encefalomielitis autoimmune experimental (EAE). De la mateixa forma la proteïna priònica cel•lular és una proteïna que s’ha associat majoritàriament a malalties espongiformes, però que recentment s’ha vinculat (no sense controvèrsia) amb la seva possible relació amb la Malaltia d'Alzheimer (AD), ja que seria capaç de reclutar els oligòmers d’Aβ (ADDLs), els quals correlacionen millor amb el grau de demència, i amb els que interacciona directament, actuant així com un possible mediador de la fosforilació de tau en la malaltia. No obstant, les funcions de les MAIS i de la PrPc en aquests models de la malaltia no estan clarament definits i, per altra banda, es desconeixen els mecanismes de senyalització implicats, no descartant de forma clara el component neural i l’immune.

Visualització de la interaccio entre la proteïnaG12 i la catenina p120

Els processos de senyalització a través de receptors acoplats a proteïnes G (GPCRs) estan implicats en una gran varietat de processos fisiològics i patològics. Els objectius de la meva recerca es centren en l’estudi de la funció de les proteïnes heterotrimèriques de la família G12, en particular, en el paper que aquestes proteïnes poden tenir en la inducció de la migració cel•lular. Des del seu descobriment, s’han descrit diversos efectors que s’uneixen i es regulen per aquestes proteïnes. Les proteïnes de la família de RhoGEF semblen ser els efectors més directes i que juguen un paper més important en els processos de senyalització de les proteïnes G12. Tanmateix, resultats recents semblen indicar que altres vies independent de l’activació de Rho són també necessàries perquè els efectes fisiològics de les vies de les proteïnes G12 tinguin lloc. En aquest camp, els resultats que he obtingut, juntament amb resultats previs del grup, han descrit una nova via d’activació independent de Rho. Hem trobat que la proteïna G12 s’uneix a una catenina: la catenina p120. La seva unió sembla tenir lloc a través l’extrem N-terminal de la catenina i condueix a la reducció de la fosforilació en algun dels seus residus de tirosina, ja sigui per la quinasa Src o per l’activació a través d’EGF. Per tant, aquests resultats suggereixen que una altra via d’acció de la proteïna G12 seria mitjançant la regulació de la catenina p120 i, com a conseqüència, alguns processos d’adhesió cel•lular es podrien veure afectats. A fi d’entendre millor la regulació i la interacció de la catenina p120 hem iniciat l’estudi de la seva distribució cel•lular en l’espai i temps mitjançant tècniques de microscopia. Així, vam intentar construir una sonda de G12 fluorescent amb GFP. Després de diversos intents i experiments amb proteïnes no funcionals hem aconseguit, amb la col•laboració de T. Meigs, una construcció funcional que activa Rho. Això ens permetrà acabar els experiments de seguiment in vivo.

Involvement of Dab1 in APP processing and [beta]-amyloid deposition in sporadic Creutzfeldt-Jakob patients.

Alzheimer"s disease and prion pathologies (e.g., Creutzfeldt-Jakob disease) display profound neural lesions associated with aberrant protein processing and extracellular amyloid deposits. For APP processing, emerging data suggest that the adaptor protein Dab1 plays a relevant role in regulating its intracellular trafficking and secretase-mediated proteolysis. Although some data have been presented, a putative relationship between human prion diseases and Dab1/APP interactions is lacking. Therefore, we have studied the putative relation between Dab1, APP processing and Aβ deposition, targets in sCJD cases. Our biochemical results categorized two groups of sCJD cases, which also correlated with PrPsc types 1 and 2 respectively. One group, with PrPsc type 1 showed increased Dab1 phosphorylation, and lower βCTF production with an absence of Aβ deposition. The second sCJD group, which carried PrPsc type 2, showed lower levels of Dab1 phosphorylation and βCTF production, similar to control cases. Relevant Aβ deposition in the second sCJD group was measured. Thus, a direct correlation between Dab1 phosphorylation, Aβ deposition and PrPsc type in human sCJD is presented for the first time.

Myosin motors and not actin comets are mediators of the actin-based Golgi-to-endoplasmic reticulum protein transport

We have previously reported that actin filaments are involved in protein transport from the Golgi complex to the endoplasmic reticulum. Herein, we examined whether myosin motors or actin comets mediate this transport. To address this issue we have used, on one hand, a combination of specific inhibitors such as 2,3-butanedione monoxime (BDM) and 1-[5-isoquinoline sulfonyl]-2-methyl piperazine (ML7), which inhibit myosin and the phosphorylation of myosin II by the myosin light chain kinase, respectively; and a mutant of the nonmuscle myosin II regulatory light chain, which cannot be phosphorylated (MRLC2AA). On the other hand, actin comet tails were induced by the overexpression of phosphatidylinositol phosphate 5-kinase. Cells treated with BDM/ML7 or those that express the MRLC2AA mutant revealed a significant reduction in the brefeldin A (BFA)-induced fusion of Golgi enzymes with the endoplasmic reticulum (ER). This delay was not caused by an alteration in the formation of the BFA-induced tubules from the Golgi complex. In addition, the Shiga toxin fragment B transport from the Golgi complex to the ER was also altered. This impairment in the retrograde protein transport was not due to depletion of intracellular calcium stores or to the activation of Rho kinase. Neither the reassembly of the Golgi complex after BFA removal nor VSV-G transport from ER to the Golgi was altered in cells treated with BDM/ML7 or expressing MRLC2AA. Finally, transport carriers containing Shiga toxin did not move into the cytosol at the tips of comet tails of polymerizing actin. Collectively, the results indicate that 1) myosin motors move to transport carriers from the Golgi complex to the ER along actin filaments; 2) nonmuscle myosin II mediates in this process; and 3) actin comets are not involved in retrograde transport.

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.