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Mechanisms of PAR-1 mediated kinase receptor transactivation: Smad linker region phosphorylation

  • Danielle KamatoEmail author
  • Hang Ta
  • Rizwana Afroz
  • Suowen Xu
  • Narin Osman
  • Peter J. Little
Research Article
  • 7 Downloads

Abstract

Protease activated receptors (PARs) transactivate both epidermal growth factor receptors (EGFR) and transforming growth factor (TGF)-β receptors (TGFBR1) in vascular smooth muscle leading to the increased expression of genes (CHST11 and CHSY1) which are rate limiting for the enzymes that mediate hyperelongation of glycosaminoglycan (GAG) chains on the lipid-binding proteoglycan, biglycan. This is an excellent model to investigate mechanisms of transactivation as the processes are biochemically distinct. EGFR transactivation is dependent on the classical matrix metalloprotease (MMP) based triple membrane bypass mechanism and TGFBR1 transactivation is dependent on Rho/ROCK signalling and integrins. We have shown that all kinase receptor signalling is targeted towards phosphorylation of the linker region of the transcription factor, Smad2. We investigated the mechanisms of thrombin mediated kinase receptor transactivation signalling using anti-phospho antibodies and Western blotting and gene expression by RT-PCR. Thrombin stimulation of phospho-Smad2 (Ser 245/250/255) and of phospho-Smad2(Thr220) via EGFR transactivation commences quickly and extends out to at least 4 h whereas transactivation via TGFBR1 is delayed for 120 min but also persists for at least 4 h. Signalling of thrombin stimulated Smad linker region phosphorylation is approximately equally inhibited by the MMP inhibitor, GM6001 and the ROCK inhibitor, Y27632, and similarly expression of CHST11 and CHSY1 is approximately equally inhibited by GM6001 and Y27632. The data establishes Smad linker region phosphorylation as a central target of all transactivation signalling of GAG gene expression and thus an upstream kinase may be a target to prevent all transactivation signalling and its pathophysiological consequences.

Keywords

Transactivation signalling G protein coupled receptors Smad Smad linker region G proteins Serine/threonine kinase receptors 

Notes

Funding

DK was supported by the NHMRC (APP1160925) and National Heart Foundation Fellowship (102129). Support was received from the University of Queensland through a personal support package to PJL and by the University of Queensland Early Career Grant (DK) (Grant no. 1832825).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

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Copyright information

© The International CCN Society 2019

Authors and Affiliations

  1. 1.School of PharmacyUniversity of QueenslandWoolloongabbaAustralia
  2. 2.Department of PharmacyXinhua College of Sun Yat-sen UniversityGuangzhouChina
  3. 3.Aab Cardiovascular Research Institute, Department of MedicineUniversity of Rochester School of Medicine and DentistryRochesterUSA
  4. 4.School of Health and Biomedical SciencesRMIT UniversityBundooraAustralia
  5. 5.Department of Immunology and PathologyMonash UniversityMelbourneAustralia

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