Nauphar, Donny and Faradz, Sultana MH and Mundhofir, Farmaditya E.P. (2013) PROFILING SERINE-THREONINE KINASE PHOSPHORYLATION IN TGFβ PATHWAY IN TAAD PATIENTS WITH TGFBR2 MUTATION. Masters thesis, Universitas Diponegoro.

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Abstract

Background: Thoracic aortic aneurysm and dissection (TAAD) is one of the top 20 most leading causes in the USA and one of the silent killers in the world. The dysregulation of TGFβ pathway has been linked with pathogenesis of the disease. TGFβ pathway is a tightly regulated pathway that is controlled by phosphorylation of their downstream secondary messenger. The kinase substrate peptide microarray is utilized to see how the phosphorylation pattern of TGFβ downstream secondary messenger is regulated in TAAD patient.
Methods: Fibroblast samples from 3 mutants with TAAD carrying TGFβRII mutations and 3 normal patients were grown and stimulated with TGF-β1after 24 hours of serum starvation. The cells were then lysed and their protein concentration determined using bicinchoninic acid (BCA) assay. The phosphorylation of kinase substrate peptides were then analyzed using Serine- Threonine Kinase Microarray Chip.
Results: Kinase substrate peptides were chosen based on the significant phosphorylation changes between controls and mutants of the stimulated and unstimulated groups. Four kinase substrate peptides were found under- phosphorylated between the controls and mutants of the TGF-β1 unstimulated group. The TGF-β1 stimulated group yields 34 significantly over phosphorylated peptides and 1 under-phosphorylated peptide.
Conclusion: The non-canonical and canonical pathway is activated
simultaneously in TAAD patients despite the absence of TGFβRII. Kinase substrate peptide has huge potential to unravel the complicated TGFβ pathway via studying the phosphorylation pattern of TGFβ downstream secondary messenger.
Keywords: Thoracic aortic aneurysm and dissection, TGF-β1, TGFβRII, Serine-Threonine Kinase, Microarray

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