Modeling and subtleties of K-Ras and Calmodulin interaction

K-Ras, one of the most common small GTPases of the cell, still presents many riddles, despite the intense efforts to unveil its mysteries. Such is the case of its interaction with Calmodulin, a small acidic protein known for its role as a calcium ion sensor. Although the interaction between these two proteins and its biological implications have been widely studied, a model of their interaction has not been performed. In the present work we analyse this intriguing interaction by computational means. To do so, both conventional molecular dynamics and scaled molecular dynamics have been used. Our simulations suggest a model in which Calmodulin would interact with both the hypervariable region and the globular domain of K-Ras, using a lobe to interact with each of them. According to the presented model, the interface of helixes α4 and α5 of the globular domain of K-Ras would be relevant for the interaction with a lobe of Calmodulin. These results were also obtained when bringing the proteins together in a step wise manner with the umbrella sampling methodology. The computational results have been validated using SPR to determine the relevance of certain residues. Our results demonstrate that, when mutating residues of the α4-α5 interface described to be relevant for the interaction with Calmodulin, the interaction of the globular domain of K-Ras with Calmodulin diminishes. However, it is to be considered that our simulations indicate that the bulk of the interaction would fall on the hypervariable region of KRas, as many more interactions are identified in said region. All in all our simulations present a suitable model in which K-Ras could interact with Calmodulin at membrane level using both its globular domain and its hypervariable region to stablish an interaction that leads to an altered signalling

Toward understanding calmodulin plasticity by molecular dynamics

Aim: Calmodulin interacts in many different ways with its ligands. We aim to shed light on its plasticity analysing the changes followed by the linker region and the relative position of the lobes using conventional Molecular Dynamics (cMD), accelerated MD (aMD) and scaled MD (sMD). Materials & Methods: Three different structures of calmodulin are compared, obtaining a total of 2.5 μs of molecular dynamics, which have been analysed using the principal component analysis and clustering methodologies Results: sMD simulations reach conformations that cMD is not able to, without compromising the stability of the protein. On the other hand, aMD requires optimization of the setup parameters to be useful. Conclusion: SMD is useful to study flexible proteins, highlighting those factors that justify its promiscuit

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

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

Ribonucleoprotein HNRNPA2B1 interacts with and regulates oncogenic KRAS in Pancreatic Ductal Adenocarcinoma Cells.

BACKGROUND & AIMS: Development of pancreatic ductal adenocarcinoma (PDAC) involves activation of c-Ki-ras2 Kirsten rat sarcoma oncogene homolog (KRAS) signaling, but little is known about the roles of proteins that regulate the activity of oncogenic KRAS. We investigated the activities of proteins that interact with KRAS in PDAC cells. METHODS: We used mass spectrometry to demonstrate that heterogeneous nuclear ribonucleoproteins (HNRNP) A2 and B1 (encoded by the gene HNRNPA2B1) interact with KRAS G12V. We used co-immunoprecipitation analyses to study interactions between HNRNPA2B1 and KRAS in KRAS-dependent and KRAS-independent PDAC cell lines. We knocked down HNRNPA2B1 using small hairpin RNAs and measured viability, anchorage-independent proliferation, and growth of xenograft tumors in mice. We studied KRAS phosphorylation using the Phos-tag system. RESULTS: We found that interactions between HRNPA2B1 and KRAS correlated with KRAS-dependency of some human PDAC cell lines. Knock down of HNRNPA2B1 significantly reduced viability, anchorage-independent proliferation, and formation of xenograft tumors by KRAS-dependent PDAC cells. HNRNPA2B1 knock down also increased apoptosis of KRAS-dependent PDAC cells, inactivated c-akt murine thymoma oncogene homolog 1 signaling via mammalian target of rapamycin, and reduced interaction between KRAS and phosphatidylinositide 3-kinase. Interaction between HNRNPA2B1 and KRAS required KRAS phosphorylation at serine 181. CONCLUSIONS: In KRAS-dependent PDAC cell lines, HNRNPA2B1 interacts with and regulates the activity of KRAS G12V and G12D. HNRNPA2B1 is required for KRAS activation of c-akt murine thymoma oncogene homolog 1-mammalian target of rapamycin signaling, interaction with phosphatidylinositide 3-kinase, and PDAC cell survival and tumor formation in mice. HNRNPA2B1 might be a target for treatment of pancreatic cancer

KRAS phosphorylation regulates cell polarization and tumorigenic properties in colorectal cancer.

Oncogenic mutations of KRAS are found in the most aggressive human tumors, including colorectal cancer. It has been suggested that oncogenic KRAS phosphorylation at Ser181 modulates its activity and favors cell transformation. Using nonphosphorylatable (S181A), phosphomimetic (S181D), and phospho-/dephosphorylatable (S181) oncogenic KRAS mutants, we analyzed the role of this phosphorylation to the maintenance of tumorigenic properties of colorectal cancer cells. Our data show that the presence of phospho-/dephosphorylatable oncogenic KRAS is required for preserving the epithelial organization of colorectal cancer cells in 3D cultures, and for supporting subcutaneous tumor growth in mice. Interestingly, gene expression differed according to the phosphorylation status of KRAS. In DLD-1 cells, CTNNA1 was only expressed in phospho-/dephosphorylatable oncogenic KRAS-expressing cells, correlating with cell polarization. Moreover, lack of oncogenic KRAS phosphorylation leads to changes in expression of genes related to cell invasion, such as SERPINE1, PRSS1,2,3, and NEO1, and expression of phosphomimetic oncogenic KRAS resulted in diminished expression of genes involved in enterocyte differentiation, such as HNF4G. Finally, the analysis, in a public data set of human colorectal cancer, of the gene expression signatures associated with phosphomimetic and nonphosphorylatable oncogenic KRAS suggests that this post-translational modification regulates tumor progression in patients

Anàlisi de la maquinària reguladora del cicle cel·lular en diferents models de proliferació

[cat] Aquesta tesi té com a principals objectius: 1- Identificació de nous substrats de cdk2 en diferents models cel·lulars: cèl·lules HeLa, hepatòcits normals activats a proliferar mitjançant una hepatectomia parcial, limfòcits humans normals activats a proliferar, i la línia limfoide Namalwa. 2- Localització intranuclear de proteïnes que participen en el control del cicle cel·lular en dos models de proliferació un normal i l'altre transformat: hepatòcits normals activats a proliferar mitjançant una hepatectomia parcial, i la línia cel·lular transformada d'origen humà HeLa. 3- Anàlisi dels mecanismes d'activació de la cdk4 i la cdk2 durant la regeneració hepàtica

Modeling and subtleties of K-Ras and Calmodulin interaction

K-Ras, one of the most common small GTPases of the cell, still presents many riddles, despite the intense efforts to unveil its mysteries. Such is the case of its interaction with Calmodulin, a small acidic protein known for its role as a calcium ion sensor. Although the interaction between these two proteins and its biological implications have been widely studied, a model of their interaction has not been performed. In the present work we analyse this intriguing interaction by computational means. To do so, both conventional molecular dynamics and scaled molecular dynamics have been used. Our simulations suggest a model in which Calmodulin would interact with both the hypervariable region and the globular domain of K-Ras, using a lobe to interact with each of them. According to the presented model, the interface of helixes α4 and α5 of the globular domain of K-Ras would be relevant for the interaction with a lobe of Calmodulin. These results were also obtained when bringing the proteins together in a step wise manner with the umbrella sampling methodology. The computational results have been validated using SPR to determine the relevance of certain residues. Our results demonstrate that, when mutating residues of the α4-α5 interface described to be relevant for the interaction with Calmodulin, the interaction of the globular domain of K-Ras with Calmodulin diminishes. However, it is to be considered that our simulations indicate that the bulk of the interaction would fall on the hypervariable region of KRas, as many more interactions are identified in said region. All in all our simulations present a suitable model in which K-Ras could interact with Calmodulin at membrane level using both its globular domain and its hypervariable region to stablish an interaction that leads to an altered signalling

Modeling and subtleties of K-Ras and Calmodulin interaction

K-Ras, one of the most common small GTPases of the cell, still presents many riddles, despite the intense efforts to unveil its mysteries. Such is the case of its interaction with Calmodulin, a small acidic protein known for its role as a calcium ion sensor. Although the interaction between these two proteins and its biological implications have been widely studied, a model of their interaction has not been performed. In the present work we analyse this intriguing interaction by computational means. To do so, both conventional molecular dynamics and scaled molecular dynamics have been used. Our simulations suggest a model in which Calmodulin would interact with both the hypervariable region and the globular domain of K-Ras, using a lobe to interact with each of them. According to the presented model, the interface of helixes α4 and α5 of the globular domain of K-Ras would be relevant for the interaction with a lobe of Calmodulin. These results were also obtained when bringing the proteins together in a step wise manner with the umbrella sampling methodology. The computational results have been validated using SPR to determine the relevance of certain residues. Our results demonstrate that, when mutating residues of the α4-α5 interface described to be relevant for the interaction with Calmodulin, the interaction of the globular domain of K-Ras with Calmodulin diminishes. However, it is to be considered that our simulations indicate that the bulk of the interaction would fall on the hypervariable region of KRas, as many more interactions are identified in said region. All in all our simulations present a suitable model in which K-Ras could interact with Calmodulin at membrane level using both its globular domain and its hypervariable region to stablish an interaction that leads to an altered signalling

RAD51 is a druggable target that sustains replication fork progression upon DNA replication stress.

Solving the problems that replication forks encounter when synthesizing DNA is essential to prevent genomic instability. Besides their role in DNA repair in the G2 phase, several homologous recombination proteins, specifically RAD51, have prominent roles in the S phase. Using different cellular models, RAD51 has been shown not only to be present at ongoing and arrested replication forks but also to be involved in nascent DNA protection and replication fork restart. Through pharmacological inhibition, here we study the specific role of RAD51 in the S phase. RAD51 inhibition in non-transformed cell lines did not have a significant effect on replication fork progression under non-perturbed conditions, but when the same cells were subjected to replication stress, RAD51 became necessary to maintain replication fork progression. Notably, the inhibition or depletion of RAD51 did not compromise fork integrity when subjected to hydroxyurea treatment. RAD51 inhibition also did not decrease the ability to restart, but rather compromised fork progression during reinitiation. In agreement with the presence of basal replication stress in human colorectal cancer cells, RAD51 inhibition reduced replication fork speed in these cells and increased γH2Ax foci under control conditions. These alterations could have resulted from the reduced association of DNA polymerase α to chromatin, as observed when inhibiting RAD51. It may be possible to exploit the differential dependence of non-transformed cells versus colorectal cancer cells on RAD51 activity under basal conditions to design new therapies that specifically target cancer cell