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

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Journal of Cell Communication and Signaling Aims and scope

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.

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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).

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Authors and Affiliations

  1. School of Pharmacy, University of Queensland, The University of Queensland, 20 Cornwall Street, Woolloongabba, QLD 4102, Australia

    Danielle Kamato, Hang Ta, Rizwana Afroz & Peter J. Little

  2. Department of Pharmacy, Xinhua College of Sun Yat-sen University, Tianhe District, Guangzhou, 510520, China

    Danielle Kamato & Peter J. Little

  3. Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA

    Suowen Xu

  4. School of Health and Biomedical Sciences, RMIT University, Bundoora, VIC, 3083, Australia

    Narin Osman

  5. Department of Immunology and Pathology, Monash University, Melbourne, VIC, 3004, Australia

    Narin Osman

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  1. Danielle Kamato
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  3. Rizwana Afroz
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  4. Suowen Xu
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Correspondence to Danielle Kamato.

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Kamato, D., Ta, H., Afroz, R. et al. Mechanisms of PAR-1 mediated kinase receptor transactivation: Smad linker region phosphorylation. J. Cell Commun. Signal. 13, 539–548 (2019). https://doi.org/10.1007/s12079-019-00527-5

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