PASCO Alto Perú, comunidad

193

Exploring the design principles of signaling circuits in space and time.

W. Lim1; 1Cellular & Molecular Pharmacology, Univ California-San Francisco/HHMI, San Francisco, CA

Living cells are able to carry out diverse information processing tasks and to control complex spatiotemporal behaviors using molecular signal transduction networks. To understand the design principles of such networks, we are complementing traditional analytical approaches with synthetic approaches of trying to build novel or systematically modified networks that can carry out target functional behaviors. Using modular components that control phosphorylation and GTPase signaling, we are exploring the construction of synthetic spatial self-organizing circuits, as well as circuits with complex dynamical responses. We are also using parallel enumeration based computational approaches to explore the space of networks that can robustly execute such tasks. We hope to gain a deeper understanding between network structure and function through such studies. We also hope to gain a better understanding of the evolutionary landscape through which such behaviors emerge.

194

Spatio-temporal control of intracellular ROS concentration in PDGF signaling revealed by single Eu3+-doped nanoparticle imaging.

C. Bouzigues1, T-L. Nguyen1, R. Ramodiharilafy1, P-L. Tharaux2, A. Alexandrou1; 1Laboratoire Optique et Biosciences, Ecole Polytechnique, Palaiseau, France, 2PARCC, HEGP, Paris, France

For many cell functions, notably those requiring an asymmetric response, such as directed migration, spatio-temporal organization of signaling pathways is important for the cell response regulation. PDGF (Platelet Derived Growth Factor) induces migration in numerous contexts, such as reparation of vascular lesions or metastasis formation, inducing ROS (Reactive Oxygen Species) production as second messenger. The potential lethality of concentrated ROS and the importance of intracellular organization for migration require a tight control of their

concentration. However, the dynamics of ROS production and organization is so far mostly unknown. By imaging single Eu3+-doped nanoparticles1, we probed the intracellular ROS response with high temporal and spatial resolution. We thus measured the absolute ROS concentration in normal or tumoral cells and revealed specific temporal patterns of ROS production under PDGF stimulation. We measured an integration time of several minutes required for the ROS response formation. This response formation is shorter for tumoral cells. We satisfyingly explained this fact by modeling the diffusion-limited dimerization of PDGFRs. This may constitute a temporal filtering mechanism for preventing cell responses to transient signals and its impairment in tumoral cells could be of great physiological relevance for the metastatic transition. We moreover quantitatively measured a transactivation of EGFR by PDGF stimulation and revealed its role in the dynamics of the cell response. By using a microfluidic system, we furthermore apply spatially controlled PDGF stimulation and displayed the

maintenance of asymmetric ROS concentration in the cell under a PDGF gradient. This likely relies on a balance between ROS diffusion and degradation. This balance controls the local ROS concentration and thus the cell response. Altogether, our results reveal the tight regulation of the ROS spatio-temporal organization by the cell, which illustrates how the spatio-temporal control of transduction pathways is crucial for the buildup of the cell response.

1_{Casanova, Bouzigues et al. Nat. Nanotech. 4, 581 (2009)}

195

Dynamics: SH2 domain binding/unbinding on EGFR.

D. Oh1, K. Machida1, M. Ogiue-Ikeda1 , J. A. Jadwin1, B. J. Mayer1, J. Yu1; 1CCAM, University of Connecticut Health Center, Farmington, CT

Epidermal growth factor (EGF) stimulation triggers downstream signaling cascades through binding of Src homology 2 (SH2) domain containing proteins to the phosphorylated tyrosine (p- Tyr) residues of the EGF receptor (EGFR). This signaling plays a crucial role in cell proliferation, metastasis, survival, tumorigenesis, etc. However the dynamics of bindings of SH2 domain to p- Tyr on the EGFR are not well quantified. Applying novel single-particle-tracking photoactivated localization microscopy (spt-PALM) techniques combined with the total internal reflection (TIR) optics, we quantified kinetic parameters for following tyrosine phosphorylation rate and dissociation rate between EGFR and tdEos- fused SH2 domains of several signaling molecules (Grb2, Plc 1, Shp2, Src, Nck1, Crk, Jak2, ShcA, CblA) in live cell. We mapped the progression of the EGFR-SH2 complexes over the EGF induction time and measured binding kinetics. Our data shows each SH2 domain produced a different binding curve as a function of time after EGF stimulation, suggesting phosphorylation rates are different for different tyrosine residues on EGFR and each SH2 domain has binding specificity. Analyzing multiple exponential distributions of an individual binding time of SH2 domain, we obtained the dissociation rate from

0.05 to 10 S-1. This finding implies that there exist multiple states of EGFR-SH2 complex such as transient, monomer or clustering binding states. The clustering of EGFR-SH2 complex developed as a function of EGF stimulation time was confirmed by tracking approximately 1-5 x104 individual complexes with the time-course PALM imaging. Interestingly, a negative correlation between binding time of SH2 domain on EGFR and their mobility was observed. This result, along with PALM analysis, suggests that downstream signaling may require stable clustering of EGFR-SH2 complexes. In conclusion, our analyses not only confirmed binding specificity of SH2 domains, but also suggested that each specific p-Tyr residue on the receptor has a specific phosphorylation rate.

196

Targeted Proteomics Analysis of Signalling Dynamics: a View of the Adaptor Protein GRB2 mediated events.

N. Bisson1, A. James1, G. Ivosev2, S. Tate2, L. Taylor1, T. Pawson1; 1Samuel Lunenfeld Res Inst, Toronto, ON, Canada, 2AB Sciex

Signalling pathways are commonly organized through inducible protein-protein interactions, mediated by adaptor proteins that link activated receptors to cytoplasmic effectors. However, we have little quantitative data regarding the kinetics with which such networks assemble and dissolve to generate a specific cellular response. We have identified 90 proteins and 36 phosphorylation sites associated with the GRB2 adaptor protein in human cells. We have found that GRB2 nucleates a remarkably diverse set of protein complexes, involved in multiple aspects of cellular function. To comprehensively and quantitatively investigate changes in GRB2-based protein interactions in growth factor stimulated cells, we have designed a targeted mass spectrometry method, AP-SRM (affinity purification-selected reaction monitoring). The data define context-specific and time-dependent networks that form around GRB2 following stimulation, and reveal core and growth factor-selective interaction subsets. These results illustrate the reliability of AP-SRM in the quantitative analysis of dynamic signalling networks. They also suggest that capturing a key hub protein and dissecting its interactions by SRM is an approach that can be broadly applied to quantify signalling dynamics.

197

KLF5 acetylation, which is induced by TGFbeta but interrupted by oncogenic signaling, underlies the dual functions of TGFbeta in gene regulation and cell proliferation control. J-T. Dong1, P. Guo1; 1Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA

During epithelial homeostasis, stem cells divide to produce progenitor cells, which not only proliferate to generate the cell mass but also respond to cellular signaling to transition from a proliferative state to a differentiation state. Such a transition involves functional alterations of transcriptional factors, yet the underlying molecular mechanisms are poorly understood. In addressing this question, we found that the pro-proliferative factor KLF5 becomes anti- proliferative upon TGFbeta-mediated acetylation in an in vitro model of epithelial homeostasis. KLF5 is not only essential for cell proliferation, it is also indispensable for TGFbeta-induced anti- proliferation in epithelial cells. Without TGFbeta, KLF5 inhibits p15 expression and induces Myc expression, but when TGF-¦Â is present, KLF5 becomes a cofactor of TGFbeta to induce p15 and suppress Myc expression in the same cells. Mechanistically, TGFbeta recruits acetylase p300 to acetylate KLF5, and acetylation in turn induces the assembly of KLF5 with other TGFbeta transcriptinoal effectors including Smad2-4 and Miz-1 and their binding to p15 and Myc promoters, resulting in the reversal of KLF5 function. Failure in KLF5 acetylation prevents TGFbeta-assemblled p300-KLF5-Smads complex on gene promoters, reversing the function of

TGFbeta in gene regulation and proliferation control. It was reported that TGFbeta and KLF5 have dual roles in tumoirigenesis, and that Ras oncogenic signaling converts TGFbeta from a tumor suppressor to a tumor promoter. We therefore also examined whether Ras signaling modulates TGFbeta function by interrupting TGFbeta-induced KLF5 acetylation and the assembly of the p300-KLF5-Smads transcriptional complex. We found that Ras inhibited TGFbeta-induced KLF5 acetylation and interfered with TGFbeta in p15 induction and Myc repression. In addition, TGFbeta-induced Smad3 phosphorylation at the C-terminal region was necessary for TGFbeta to induce KLF5 acetylation, and Ras interrupted this phosphorylation. Ras signaling further interrupted the interactions among p300, KLF5 and Smads, as well as the binding of the p300-KLF5-Smads complex onto the TGFbeta-responsive promoter elements for both p15 and Myc. Our findings suggest that 1) TGFbeta-induced KLF5 acetylation is a "switch" that that turns on differentiation in proliferating progenitor cells; 2) deacetylation of KLF5 underlies the reversal of TGFbeta function in gene regulation and cell proliferation control; and 3) oncogenic signaling reverses TGFbeta function by interrupting KLF5 acetylation.

198

Cross-talk and information transfer in mammalian and bacterial signaling.

S. Lyons1, A. Prasad2; 1School of Biomedical Engineering, Colorado State University, Fort Collins, CO, 2Chemical and Biological Engineering, Colorado State University, Fort Collins, CO In both mammalian cells and bacteria, simple phosphorylation circuits play a very important role in cellular function. Bacteria have hundreds of two-component signaling systems that involve phosphotransfer between a receptor kinase and a response regulator. In mammalian cells a similar pathway is the crucial TGF-beta signaling pathway, where extracellular levels of TGF- beta family ligands lead to activation of cell surface receptors that phosphorylate Smad proteins, which in turn activate many genes. In TGF-beta signaling the multiplicity of external ligands begs the question as to how cells are able to distinguish signals coming from different extra- cellular ligands, but transduced through a small set of Smads. Here we use information theory with stochastic simulations of simple networks to address this question. We find that when signals are transduced through the same Smad, the cell cannot distinguish between different levels of the external ligands. Increasing the number of Smads from one to two significantly improves information transmission as well as the ability to discriminate between different external ligands. Surprisingly, both total information transmitted through the channel and the capacity to discriminate between the external ligands are quite insensitive to the cross-talk between the two Smads as long as they are not nearly identical. In sharp contrast, we find that two-component systems in bacteria show a significantly sharper decline in information transfer in the presence of cross-talk. This suggests that mammalian signal transduction can tolerate a high amount of cross-talk. This may have played a role in the evolution of new functionalities from small mutations in signaling pathways and allowed for the development of cross-regulation. Insensitivity to cross-talk also could increase robustness due to redundancy in signaling pathways. On the other hand, bacterial two component systems are much less robust against cross-talk which may provide an explanation for the lack of cross-regulation in most two component systems.