Estudio de la Interacción entre la Proteína Asociada a Microtúbulos 1B y la Enzima Tirosina Tubulina Ligasa en Neuronas

Memoria para optar el título de BioquímicoLas modificaciones post-traduccionales de la α-tubulina contribuyen a determinar las propiedades dinámicas de ciertas poblaciones de microtúbulos. Esto es particularmente importante para procesos que requieren cambios rápidos en la estabilidad del citoesqueleto que permitan la modificación de las propiedades morfológicas o motiles de las neuronas. Estas modificaciones deberían regularse localmente durante los procesos de elongación axonal, migración neuronal y guía axonal. La proteína asociada a microtúbulos 1 B (MAP1B) es una proteína que participa y regula los tres procesos antes mencionados. Se ha descrito que la presencia de una variante fosforilada de MAP1B está enriquecida en el extremo distal de axones, esencialmente la zona más dinámica del axón. Se ha descrito que conjuntamente con el gradiente de MAP1B fosforilada, se produce un gradiente de microtúbulos alfa-tirosinados. Esta modificación post-traduccional es efectuada por acción de la enzima tubulina tirosina ligasa (TTL) . La hipótesis planteada en nuestro trabajo es: hipótesis: La proteína asociada a microtúbulos 1B (MAP1B) interactúa con la enzima Tubulina Tirosina Ligasa (TTL) aumentando de esta forma la tirosinación de los microtúbulos neuronales . Nuestros resultados indican que MAP1B y TTL interaccionan, evidenciado mediante ensayos de inmunoprecipitación y pull-down. Adicionalmente se muestra que la sobre-expresión regulada de la enzima glicógeno sintasa quinasa 3 beta (Gsk3ß) en neuroblastomas murinos (N2a), induce cambios en el patrón de fosforilación de MAP1B y que esta modificación no produce cambios en la interacción entre ambas proteínas. La interacción de ambas proteínas es independiente de un entrecruzamiento originado por los microtúbulos y se produce con la cadena pesada de la MAP1B. En base a estos resultados se espera comprender de mejor forma como la modificación post-traduccional de los microtúbulos, particularmente la adición de un residuo de tirosina en el extremos carboxilo terminal de la α-tubulina, juega un papel importante en procesos como migración, guía y/o elongación axonal de las neuronasPost-translational modifications of α-tubulin contribute to determine the dynamic properties of certain microtubule populations. This is particularly important in processes that require fast changes in the cytoskeleton stability to allow the modifications of the motility and morphologic properties of neurons. These modifications must be regulated locally during axonal elongation, neuronal migration and axonal guidance process. The Microtubule Associated Protein 1B (MAP1B) participates and regulates the three processes mentioned above. It has been described that the presence of a variant phosphorylated form of MAP1B is enriched in the distal end of axons, essentially in the most dynamic regions of the axon. Interestingly, it has been described that together with the gradient of phosphorilated MAP1B, a gradient of tyrosinated microtubules takes place. This posttranslational modification is carried out by the action of the tubulin tyrosin ligase (TTL) enzyme. The hypothesis proposed in our work is: The Microtubule Associated Protein 1B (MAP1B) interact with the tubulin tyrosin ligase (TTL) enzyme regulating the tyrosination of neural microtubules . Our results indicate that MAP1B and TTL interact; this was studied by inmmunoprecipitation and pull-down experiments. Additionally we showed that the regulated over expression of the glycogen synthase kinase 3 beta (Gsk3ß) enzyme in murine neuroblastoma (N2a), induces changes in the phosphorilation patterns of MAP1B and that this modification do not produces changes in the interaction between these proteins. The interaction of both proteins is independent of a crosslinking originated by the microtubules and it ocurrs with the heavy chain of MP1B. Based on these results we expect to understand better how the post-translational modifications of microtubules, particularly the addition of a tyrosine residue in the Cterminal domain of the α-tubulin, play a important role in processes like neuronal migration, axonal elongation and axonal guidanc

Cdk5 regulates Rap1 activity

Rap1 signaling is important for migration, differentiation, axonal growth, and during neuronal polarity. Rap1 can be activated by external stimuli, which in turn regulates specific guanine nucleotide exchange factors such as C3G, among others. Cdk5 functions are also important to neuronal migration and differentiation. Since we found that pharmacological inhibition of Cdk5 by using roscovitine reduced Rap1 protein levels in COS-7 cells and also C3G contains three putative phosphorylation sites for Cdk5, we examined whether the Cdk5-dependent phosphorylation of C3G could affect Rap1 expression and activity. We co-transfected C3G and tet-OFF system for p35 over-expression, an activator of Cdk5 activity into COS-7 cells, and then we evaluated phosphorylation in serine residues in C3G by immunoprecipitation and Western blot. We found that p35 over-expression increased C3G-serine-phosphorylation while inhibition of p35 expression by tetracycline or inhibition of Cdk5 activity with roscovit

Microtubule-associated protein 1B interaction with tubulin tyrosine ligase contributes to the control of microtubule tyrosination

Microtubule-associated protein 1B (MAP1B) is the first microtubule-associated protein to be expressed during nervous system development. MAP1B belongs to a large family of proteins that contribute to the stabilization and/or enhancement of microtubule polymerization. These functions are related to the control of the dynamic properties of microtubules. The C-terminal domain of the neuronal α-tubulin isotype is characterized by the presence of an acidic polypeptide, with the last amino acid being tyrosine. This tyrosine residue may be enzymatically removed from the protein by an unknown carboxypeptidase activity. Subsequently, the tyrosine residue is again incorporated into this tubulinby another enzyme, tubulin tyrosine ligase, to yield tyrosinated tubulin. Because neurons lacking MAP1B have a reduced proportion of tyrosinated microtubules, we analyzed the possible interaction between MAP1B and tubulin tyrosine ligase. Our results show that these proteins indeed interact and that the interaction is not affected by MAP1B phosphorylation. Additionally, neurons lacking MAP1B, when exposed to drugs that reversibly depolymerize microtubules, do not fully recover tyrosinated microtubules upon drug removal. These results suggest that MAP1B regulates tyrosination of α-tubulin in neuronal microtubules. This regulation may be important for general processes involved in nervous system development such as axonal guidance and neuronal migration.This work was supported by grants from DID I03/02-2, Fundacion Andes C1406012, Fondecyt 1060040 and CSIC 21 05/06 to C.G.-B., Fondecyt to R.B.M., and by grants from the Spanish Ministry of Education and Science, and Ministry of Health to J.A.Peer reviewe

Microtubule-associated protein 1B interaction with tubulin tyrosine ligase contributes to the control of microtubule tyrosination

Microtubule-associated protein 1B (MAP1B) is the first microtubule- associated protein to be expressed during nervous system development. MAP1B belongs to a large family of proteins that contribute to the stabilization and/or enhancement of microtubule polymerization. These functions are related to the control of the dynamic properties of microtubules. The C-terminal domain of the neuronal α-tubulin isotype is characterized by the presence of an acidic polypeptide, with the last amino acid being tyrosine. This tyrosine residue may be enzymatically removed from the protein by an unknown carboxypeptidase activity. Subsequently, the tyrosine residue is again incorporated into this tubulinby another enzyme, tubulin tyrosine ligase, to yield tyrosinated tubulin. Because neurons lacking MAP1B have a reduced proportion of tyrosinated microtubules, we analyzed the possible interaction between MAP1B and tubulin tyrosine ligase. Our results show that these proteins indeed interact and that the

Searching for novel Cdk5 substrates in brain by comparative phosphoproteomics of wild type and Cdk5-/- mice.

Protein phosphorylation is the most common post-translational modification that regulates several pivotal functions in cells. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase which is mostly active in the nervous system. It regulates several biological processes such as neuronal migration, cytoskeletal dynamics, axonal guidance and synaptic plasticity among others. In search for novel substrates of Cdk5 in the brain we performed quantitative phosphoproteomics analysis, isolating phosphoproteins from whole brain derived from E18.5 Cdk5+/+ and Cdk5-/- embryos, using an Immobilized Metal-Ion Affinity Chromatography (IMAC), which specifically binds to phosphorylated proteins. The isolated phosphoproteins were eluted and isotopically labeled for relative and absolute quantitation (iTRAQ) and mass spectrometry identification. We found 40 proteins that showed decreased phosphorylation at Cdk5-/- brains. In addition, out of these 40 hypophosphorylated proteins we characterized two proteins, :MARCKS (Myristoylated Alanine-Rich protein Kinase C substrate) and Grin1 (G protein regulated inducer of neurite outgrowth 1). MARCKS is known to be phosphorylated by Cdk5 in chick neural cells while Grin1 has not been reported to be phosphorylated by Cdk5. When these proteins were overexpressed in N2A neuroblastoma cell line along with p35, serine phosphorylation in their Cdk5 motifs was found to be increased. In contrast, treatments with roscovitine, the Cdk5 inhibitor, resulted in an opposite effect on serine phosphorylation in N2A cells and primary hippocampal neurons transfected with MARCKS. In summary, the results presented here identify Grin 1 as novel Cdk5 substrate and confirm previously identified MARCKS as a a bona fide Cdk5 substrate

Putative Cdk5-dependent phosphorylation sites downregulated in Cdk5^−/− brains.

<p>In <b>bold</b> type phosphorylated sites previously described in other mouse brain phosphoproteomic studies but lacking an assigned protein kinase responsible for such a phosphorylation.</p>a<p>: phosphorylated by Cdk5,</p>b<p>: phosphorylated by Cdk1;</p>c<p>: phosphorylated by Cdk2.</p><p>PSSM scores determined as Borquez et al, 2013. NetphosK scores calculated using public protein prediction tool.</p><p>Asterisks (*) indicated Cdk5 is the best kinase for a given site.</p><p>Candidates validated in the present study are presented in italics type.</p

Cdk5 phosphorylates Grin1 at Ser369.

<p>A) Grin1 alignment showing Ser369 and Ser691 sequence as Cdk5 motifs. The shaded boxes show conserved amino acids, bold amino acid is the phosphorylation site. B) Detection of Grin1 in rat B104 and mouse N2A neuroblastoma cells and mouse brain. C) Immunoprecipitation of Grin1 and Western blot detection of Cdk5 and Grin1 in N2A cells and mouse brain. D) Immunoprecipitation of Cdk5 and Western blot detection of Grin1 and Cdk5 in N2A cells and mouse brain. E) Immunoprecipitation of Grin1 and Western blot detection of Grin1 and phospho Serine (using an antibody that recognize phosphorylation on SPXK) brain of Cdk5^+/+ and Cdk5^−/− mice. F) Immunoprecipitation of Grin1 and Western blot detection of phospho serine and Grin1 in N2A cells transfected with pBI-p35/EGFP and tTA, and pBI-p35/EGFP and tTA plus roscovitine. All data are presented as mean and SEM (<i>n</i> = 3). * p<0.05, ** p<0.01 (t-Test).</p

Proposed model illustrating potential roles of phosphorylated Grin1 by Cdk5.

<p>A) Phosphorylation of MARCKS by Cdk5 could modulate its interaction with actin filaments leading to stabilization of actin cytoskeleton. B) Involvement of Grin1 phosphorylation by Cdk5 in actin dynamics and neurite outgrowth. GPCR stimulation activates MAPK signaling pathway with increased of Egr1 and p35 expressions and subsequent increases in Cdk5 activity, which in turn phosphorylate Grin1. Additionally, GPCR stimulation promotes neurite outgrowth possibly mediated by the phosphorylation of Grin1 by Cdk5 and Cdc42-PAK-LimK-Cofilin pathway.</p