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Updated: Oct 7, 2026
Quantifying Agonist Activity at G Protein-coupled Receptors
Published on: December 26, 2011
Thiophosphorylation-induced Ca(2+) sensitization of guinea-pig ileum contractility is not mediated by Rho-associated
G Pfitzer1, D Sonntag-Bensch, D Brkic-Koric
1Department of Physiology and Pathophysiology, University of Cologne, Robert Koch-Strasse 39, D-50931 Cologne, Germany. gabriele.pfitzer@uni-koeln.de
Abstract:
1. Incubation of beta-escin-permeabilized guinea-pig longitudinal ileal smooth muscle with ATP gamma S under conditions that do not lead to thiophosphorylation of regulatory light chains of myosin (r-MLC) increased subsequent Ca(2+) sensitivity of force and r-MLC phosphorylation. In this study we tested whether this is due to activation of the Rho and/or Rho-associated kinase (ROK) as it is the case in agonist-induced Ca(2+) sensitization. 2. The increase in Ca(2+) sensitivity induced by pretreatment with ATP gamma S at pCa > 8 with the myosin light chain kinase (MLCK) inhibitor ML-9 in rigor solution was associated with (35)S incorporation into the regulatory subunit of myosin light chain phosphatase (MLCP), MYPT1, and several other high molecular mass proteins. No thiophosphorylation of r-MLC, MLCK, caldesmon, calponin and CPI-17 was detected. 3. While the relatively specific inhibitor of ROK, Y 27632, inhibited the carbachol-induced increase in Ca(2+) sensitivity with an IC(50) of 1.4 microM, the ATP gamma S-induced increase in Ca(2+) sensitivity and thiophosphorylation of MYPT1 was not inhibited. Inhibiton of Rho by exoenzyme C3 also had no effect. 4. Only staurosporine (2 microM), but not the PKC inhibitor peptide 19-31, nor genistein nor PD 98059, inhibited the ATP gamma S-induced Ca(2+) sensitization of force, r-MLC phosphorylation, and the (35)S incorporation into MYPT1. 5. The staurosporine-sensitive kinase(s) appeared to be tightly associated with the contractile apparatus because treatment of Triton-skinned preparations with ATP gamma S also induced a staurosporine-sensitive increase in Ca(2+) sensitivity of contraction. Since there was very little immunoreactivity with antibodies to p(21)-associated kinase (PAK) in Triton-skinned preparations, the staurosporine-sensitive kinase most probably is not PAK. 6. GTP gamma S had an additive effect on ATP gamma S-induced sensitization at saturating concentrations of ATP gamma S. The additional effect of GTP gamma S was inhibited by Y 27632. 7. We conclude that treatment with ATP gamma S under ATP-free conditions, unmasks a staurosporine-sensitive kinase which induces a large increase in Ca(2+) sensitivity that is most likely to be due to thiophosphorylation of MYPT1. The kinase is distinct from ROK. The physiological significance of this kinase, which is tightly associated with the contractile apparatus, is not known at present.
Insights
ATP gamma S treatment increases smooth muscle Ca(2+) sensitivity by activating a novel staurosporine-sensitive kinase, distinct from Rho-associated kinase (ROK). This kinase likely thiophosphorylates MYPT1, enhancing muscle contraction.
Area of Science:
- Biochemistry
- Physiology
- Molecular Biology
Background:
- Smooth muscle contraction is regulated by Ca(2+) sensitivity and myosin light chain phosphorylation.
- Agonist-induced Ca(2+) sensitization involves Rho and Rho-associated kinase (ROK).
- ATP gamma S can increase Ca(2+) sensitivity without directly phosphorylating myosin regulatory light chains (r-MLC).
Purpose of the Study:
- To investigate if Rho/ROK mediate the Ca(2+) sensitization induced by ATP gamma S.
- To identify the kinase responsible for ATP gamma S-induced Ca(2+) sensitization and MYPT1 thiophosphorylation.
- To determine the location and potential identity of the novel kinase.
Main Methods:
- Beta-escin-permeabilized guinea-pig ileal smooth muscle incubation with ATP gamma S.
- Measurement of Ca(2+) sensitivity, r-MLC phosphorylation, and protein thiophosphorylation using (35)S.
- Inhibition studies using ROK inhibitor Y 27632, Rho inhibitor exoenzyme C3, and various kinase inhibitors including staurosporine.
- Experiments with Triton-skinned muscle preparations and GTP gamma S.
Main Results:
- ATP gamma S induced Ca(2+) sensitization and MYPT1 thiophosphorylation, but not r-MLC thiophosphorylation.
- ROK inhibition (Y 27632) and Rho inhibition (exoenzyme C3) did not affect ATP gamma S-induced effects.
- Staurosporine, but not other tested inhibitors, blocked ATP gamma S-induced Ca(2+) sensitization, r-MLC phosphorylation, and MYPT1 thiophosphorylation.
- The staurosporine-sensitive kinase activity was associated with the contractile apparatus and distinct from PAK.
Conclusions:
- ATP gamma S unmasks a staurosporine-sensitive kinase, distinct from ROK, that increases Ca(2+) sensitivity.
- This kinase likely acts via MYPT1 thiophosphorylation, contributing to smooth muscle contraction.
- The physiological role of this novel kinase tightly associated with the contractile apparatus remains to be elucidated.
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