Applying correlation spectroscopy to explore cell signalling via dynamic analysis of plasma membrane receptors

In a multicellular organism, all the processes of cell growth, proliferation, movement and apoptosis are strictly regulated by cell signalling. Individual cells receive signals from the environment and from other cells, and adjust their behaviour accordingly. These signals are received by receptor proteins, most of which span the cell membrane and provide a way for signals from outside the cell to cause changes inside the cell. The tyrosine kinases superfamily is a representative group of such transmembrane receptors and epidermal growth factor (EGF) receptor (EGFR) is one of its most studied members. When EGF binds to EGFR, the receptor dimerizes and its intracellular kinase domain is activated. This dimerization process is crucial for initiation of signal transduction: once the kinase domain is activated it can phosphorylate tyrosine residues in C-terminus of EGFR to provide binding sites for downstream signalling proteins which transduce the signal further. In this project, we have used raster image correlation spectroscopy (RICS) supported with number and brightness analysis (N&B) to continuously monitor the EGFR monomer-dimer equilibrium in the plasma membrane of living cells. EGFR not only dimerized upon EGF challenging, but the ratio of monomer to dimer oscillated with the periodicity of about 2.5 min suggesting the dimerization is controlled by a negative feedback loop. In chapter 2.1 we investigated this negative feedback that monomerized EGFR to conclude that the process relies on the activity of several downstream signaling proteins: phospholipase Cγ, protein kinase C, and protein kinase D (PKD), while being independent of Ca2+ signaling and endocytosis. Phosphorylation of two threonine residues (T654 and T669) in juxtamembrane domain of EGFR was identified to shift the monomer-dimer equilibrium of ligand bound, active EGFR back to monomeric state. The dimerization state of the receptor correlated with the activity of downstream signaling effectors. Based on these observations, we propose a novel, negative feedback mechanism that regulates EGFR signaling via receptor monomerization. In chapter 2.2, we combined RICS with phosphotyrosine immunofluorescence and live cell imaging of downstream signaling events (Ca2+ signaling and activity of extracellular signal-regulated kinase, ERK), to further investigate structural aspects of EGFR dimerization: hydrophobic interface between receiver and activator kinase domains, and latch formation between receiver juxtamembrane domain and activator kinase domain. We confirmed that hydrophobic interface is crucial for EGFR dimerization, as well as for phosphorylation of tyrosine residues at the C-terminus and signal transduction via Ca2+ and ERK signaling pathways. The kinase activity was crucial for EGFR dimerization and signaling, and the autophosphorylation of Y954 residue in the latch binding region of the activator kinase domain facilitated asymmetric dimer formation and increased signaling strength. On the other hand, feedback phosphorylation of the T669 in the juxtamembrane latch decreased the fraction of the dimer along with decreasing the signaling strength. In summary, this research project provides an insight into EGFR dimerization process which is regulated by the autophosphorylation and feedback phosphorylation at the latch forming region and strongly correlates with the output of PLCγ-inositol-1,4,5-trisphosphate-Ca2+ and Ras-Raf-MEK-ERK signaling pathways.Summary 3 Samenvatting 5 1 General Introduction 7 1.1 Epidermal Growth Factor Signalling 7 1.1.1 The Epidermal Growth Factor Receptor and the ErbB Family 7 1.1.2 The Structure of EGFR 9 1.1.3 Asymmetric Dimer Formation as a Crucial Step in EGFR Kinase Activation 11 1.1.4 The Signalling Pathways Triggered by EGFR Activation 13 1.1.5 The Regulation of EGFR Signalling 17 1.1.6 The Role of EGFR Signalling in Cancer 20 1.2 The Aim of the Study 23 1.3 Principles of Fluorescence Imaging and Fluorescence Fluctuation Spectroscopy Techniques 24 1.3.1 Fluorescent Proteins for Labelling of the Proteins of Interest 24 1.3.2 Confocal Microscopy 27 1.3.3 Basics of the Fluorescence Fluctuation Spectroscopy 30 1.3.4 Introduction to Raster Image Correlation Spectroscopy 35 1.3.5 The Advantages and Disadvantages of Raster Image Correlation Spectroscopy 40 1.3.6 The Concept of Number & Brightness Analysis 44 Results: 2 Feedback Inhibition of EGFR Dimerization as Negative Regulator of Signalling Pathway 51 2.1.1 Introduction 52 2.1.2 Experimental Procedures 54 2.1.2.1 Plasmid construction 54 2.1.2.2 Cell culture 56 2.1.2.3 Confocal image acquisition 56 2.1.2.4 Ca2+ imaging 57 2.1.2.5 Observation of EGF binding 57 2.1.2.6 Functional imaging of extracellular signal-regulated kinase activity 58 2.1.2.7 Evaluation of Y1173 phosphorylation by immunostaining 59 2.1.2.8 Raster image acquisition 60 2.1.2.9 Raster Image Correlation Spectroscopy 60 2.1.2.10 Number and Brightness analysis 61 2.1.3 Results 63 2.1.3.1 The EGFR monomer-dimer equilibrium shifts repetitively 63 2.1.3.2 Periodical change of monomer to dimer ratio occurs in the plasma membrane, independently from endocytosis 67 2.1.3.3 Phospholipase Cγ participates in EGFR monomerization 69 2.1.3.4 PKC counteracts EGFR dimerization 74 2.1.3.5 Protein kinase D activation is essential for EGFR monomerization 75 2.1.3.6 EGFR juxtamembrane domain phosphorylation is crucial for monomerization 76 2.1.3.7 ERK activity is attenuated by EGFR feedback monomerization 78 2.1.4 Discussion 80 3 Regulation of EGFR Dimerization and Signal Strength by Tyrosine and Threonine Phosphorylation at Juxtamembrane Latch Interface 86 3.1.1 Introduction 88 3.1.2 Experimental Procedures 90 3.1.2.1 Plasmid construction 90 3.1.2.2 Cell culture 90 3.1.2.3 RICS and N&B analysis 90 3.1.2.4 Evaluation of tyrosine phosphorylation by immunostaining 91 3.1.2.5 Ca2+ imaging 91 3.1.2.6 Functional imaging of ERK activity 92 3.1.3 Results 93 3.1.3.1 Hydrophobic asymmetric dimer interface 93 3.1.3.2 Autophosphorylation at latch-binding domain 98 3.1.3.3 Feedback threonine phosphorylation at latch region 102 3.1.4 Discussion 103 4 General Discussion and Future Perspectives 107 5 List of Symbols 118 6 List of Abbreviations 120 7 References 122status: publishe