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Mechanisms of PAR-1 mediated kinase receptor transactivation: Smad linker region phosphorylation
Danielle Kamato1,2, Hang Ta3, Rizwana Afroz3
1School of Pharmacy, University of Queensland, The University of Queensland, 20 Cornwall Street, Woolloongabba, QLD 4102, Australia. d.kamato@uq.edu.au.
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.
Insights
Protease-activated receptors (PARs) activate epidermal growth factor (EGFR) and transforming growth factor-beta (TGFBR1) receptors, increasing glycosaminoglycan (GAG) chain synthesis. Smad2 linker phosphorylation is a key target for inhibiting this transactivation signaling.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- Protease-activated receptors (PARs) play a role in vascular smooth muscle cell signaling.
- PARs can transactivate epidermal growth factor receptors (EGFR) and transforming growth factor-beta receptors (TGFBR1).
- This transactivation influences the expression of genes involved in glycosaminoglycan (GAG) chain synthesis, specifically CHST11 and CHSY1, impacting biglycan proteoglycans.
Purpose of the Study:
- To investigate the distinct mechanisms of EGFR and TGFBR1 transactivation mediated by PARs in vascular smooth muscle cells.
- To identify the central signaling pathways and molecular targets involved in thrombin-induced transactivation.
- To explore the potential of targeting upstream kinases for therapeutic intervention.
Main Methods:
- Utilized anti-phospho antibodies and Western blotting to detect phosphorylated Smad2 (pSmad2) at specific sites (Ser 245/250/255 and Thr220).
- Assessed gene expression of CHST11 and CHSY1 using reverse transcription-polymerase chain reaction (RT-PCR).
- Employed matrix metalloproteinase (MMP) inhibitor (GM6001) and Rho-kinase (ROCK) inhibitor (Y27632) to elucidate signaling pathways.
Main Results:
- Thrombin stimulation led to rapid EGFR-mediated pSmad2 (Ser 245/250/255) and delayed TGFBR1-mediated pSmad2 (Thr220) signaling, both persisting for at least 4 hours.
- Both EGFR and TGFBR1 transactivation pathways were significantly inhibited by GM6001 and Y27632.
- Inhibition of MMPs and ROCK signaling equally reduced CHST11 and CHSY1 gene expression.
- Smad2 linker region phosphorylation was identified as a central convergence point for all transactivation signaling pathways.
Conclusions:
- Smad2 linker region phosphorylation is a critical downstream target of both EGFR and TGFBR1 transactivation signaling pathways.
- Both MMP-dependent and Rho/ROCK-dependent pathways converge on Smad2 phosphorylation.
- Targeting upstream kinases that regulate Smad2 linker phosphorylation could offer a unified strategy to inhibit PAR-mediated GAG gene expression and its pathological consequences.
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