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MEK1 binds βarrestin1 directly, influencing both its phosphorylation by ERK and the timing of its isoprenaline-stimulated internalization.

Meng, Dong (2010) MEK1 binds βarrestin1 directly, influencing both its phosphorylation by ERK and the timing of its isoprenaline-stimulated internalization. PhD thesis, University of Glasgow.

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Abstract

cAMP is a well studied second messenger that is ubiquitously expressed in mammals. It conducts its function by activating its downstream effectors: protein kinase A (PKA), exchange proteins regulated by cAMP (EPAC) and cyclic nucleotide gated ion-channels. The sole mechanism to inactivate cAMP is through degradation via cyclic-phosphodiesterases (PDEs). The PDEs, especially PDE4s, are involved in many diseases including asthma, chronic obstructive pulmonary disease (COPD) and depression. Therefore, PDEs have been a consistently popular research subject for decades and pharmaceutical companies have devoted considerable effort in developing PDE inhibitors. β-arrestin interacts with PDE4D5 and is a multifunctional protein that plays pivotal roles in signal transduction. It functions as an adaptor protein in the c-Raf/MEK/ERK cascade by interacting with c-Raf and ERK. In this study, I have shown that (1) β-arrestin1 can bind MEK1 directly, mediated at least in part by D26D29 of β-arrestin1 and R47K48R49 of MEK1. (2) Disruption of this association by mutagensis or small peptides decreases the phosphorylation of Ser412 on β-arrestin1, and accelerates isoprenaline-stimulated G-protein coupled receptor (GPCR) internalization. Dimerization of PDEs is considered important for their specificity. In this study, yeast two hybrid and co-immunoprecipitation were utilised to demonstrate that (1) PDE4D5 can form stable homodimers in both yeast and mammalian cell lines. (2) The dimerisation requires multiple interaction sites such as R173/N174/N175, E228/T229/L230, L306/M307/H308 and K323/T324/E325 (together named QUAD) in which R173/N174/N175 contributes most toward the dimerisation. (3) Association of an ion-pair R499/D463 also proved to be a necessary condition for dimer formation. (4) PDE4D5 dimerisation can be affected by challenge with the PDE inhibitor, anisomycin, and cAMP elevating agonists forskolin (Fsk) and isobutylmethylxanthine (IBMX). RACK1, another PDE4D5 binding partner, mediates and initiates cell migration in many cell types and affects the activity of the c-Jun NH2-terminal kinase (JNK) signalling pathway, via its interaction with PKC. SUMOylation of proteins is an important method of regulation. In the current study, preliminary investigations were undertaken to determine whether RACK1 is SUMOylated. SUMOylation of K271 of a 25-mer peptide sequence from RACK1 was observed, yet there was no SUMOylation of RACK1 observed in HEK293 cells in the presence or absence of overexpressed E3 ligases.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: beta-arrestin, MEK1, peptide array, beta2 adrenergic receptor, PDE4D5, RACK1, SUMOylation
Subjects: Q Science > QH Natural history > QH301 Biology
Q Science > QD Chemistry
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Supervisor's Name: Houslay, Prof. M.D.
Date of Award: 2010
Depositing User: Mr D Meng
Unique ID: glathesis:2010-1614
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 16 Apr 2010
Last Modified: 26 Feb 2013 11:03
URI: http://theses.gla.ac.uk/id/eprint/1614

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