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Project

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.

Date:1 Mar 2009 →  20 Jun 2017
Keywords:Cell Signalling, Fluorescence, Biochemistry
Disciplines:Biochemistry and metabolism, Systems biology, Medical biochemistry and metabolism
Project type:PhD project