Figure 1.
The RSK inhibitor BI-D1870 inhibits the contractile response to high [K+] stimulation of intact rabbit pulmonary artery.
High [K+] (179 mM) stimulation was carried out in the presence of 25 nM, 100 nM and 1 µM BI-D1870 and on paired muscle strips the corresponding concentrations of the diluent DMSO were used as indicted. 1 µM BI-D1870 significantly inhibited both the phasic and tonic component of contraction.
Figure 2.
Inhibition of ROCK does not inhibit the ability of the RSK inhibitor BI-D1870 to suppress the high [K+] contraction and BI-D1870 significantly suppresses U46619-induced force in intact pulmonary artery.
(A) Pretreatment of intact SM with the ROCK inhibitor, Y-27632 or Y-27632 plus BI-D1870 prior to stimulation with U46619. Force responses were normalized to the KCL-induced contraction taken as 100%. (B) Pretreatment of intact SM with 25 nM, 100 nM and 1 µM BI-D1870 prior to stimulation of U46619.
Figure 3.
RSK and PDK1 contribute to Ca2+-induced force, MYPT1 and RLC20 phosphorylation in α-toxin permeabilized rabbit pulmonary artery SM.
RSK inhibitor, BI-D1870 is without effect on the actomyosin ATPase. (A) RSK inhibitor, BI-D1870 (100nM), right shifted the pCa2+-force relationship and decreased the maximal force (milli-Newtons, mN) at each intracellular Ca2+ concentration (pCa) compared to the Control diluent, DMSO. (B) BI-D1870 (100 nM) decreased phosphorylation of MYPT1 Thr853 (n = 3) and RLC20 phosphorylation at Ser19 (n = 4) at maximal Ca2+-induced (pCa 4.5) force. (C) BI-D1870 (100 nM) was without effect on the ATP-induced rate of force development in Triton permeabilized mouse ileum SM with pre-thiophosphorylated light chains and depleted of ATP. T1/2 = 4.8±1.2 sec vs 5.0±1.3 sec in the Control (DMSO) vs BI-D1870 respectively, p = ns, n = 3. Thus, the inhibitor effects of BI-D18670 are not due to inhibition of the actomyosin ATPase. (D) The PDK1 kinase inhibitor GSK 2334470 (30 µM) also suppressed the pCa2+-force relationship and reduced the absolute force (mN).
Figure 4.
RSK functions as a Ca2+ independent kinase to increase force and RLC20 phosphorylation.
RSK inhibition increases the time to the onset of force and the rate of force development (t1/2) induced by the MLCP (PP1C) inhibitor microcystin-LR (10 µM) in the absence of Ca2+(pCa 9.0) The relative phosphorylation of RLC20, measured at 25 min following the addition of microcystin, was decreased in the presence of BI-D1870 (1 µM).
Figure 5.
The RSK inhibitor BI-D1870 does not function through inhibition of ZIP kinase.
The elution of full length GFP-ZIPK off of γ phosphate linked ATP resin in the presence of 100 mM ATP, 50µM BI-D1870 or blank buffer (2% DMSO) is shown. We used 100 mM ATP because of the dense resin concentration (10 mM). 100 mM ATP gives a maximal signal. The small signal given by BI-D1870 above background (2% DMSO) is not considered relevant.
Figure 6.
RSK and MEK/ERK signaling pathways contribute to TXA2-induced Ca2+ sensitized force and MYPT1 and RLC20 phosphorylation in α-toxin permeabilized rabbit pulmonary artery SM.
(A) U46619 (300 nM) induced Ca2+ sensitized force is suppressed in the presence of RSK inhibitor, BI-D1870 (100 nM). Subsequent addition of GTPγS, as a measure of the remaining component of Ca2+ sensitized force, reached the same magnitude of force but was proportionately greater in the presence of the RSK inhibitor BI-D1870 than in its absence. (B) Summary of panel A along with an effect of PDK1 kinase inhibitor GSK2334470 (30 µM) on U46619-induced force. (C) RSK inhibitor, BI-D1870 (1 µM) significantly inhibited the U46619 (300 nM) increased phosphorylation of MYPT1 Thr853 (n = 3), RLC20 phosphorylation at Ser19 (n = 5) and MYPT1 Thr696 phosphorylation (n = 5). (D) Unphosphorylated (0P), singly (1P) and doubly phosphorylated (2P) RLC20 from SM samples stimulated with U46619 with and without BI-D1870 as in panel C were separated on urea gels. A positive control for doubly phosphorylated RLC20 was a β-escin treated SM stimulated with microcystin. (E) U46619 stimulation increased the phosphorylation of proteins that regulate RSK activation; RSK2 Ser227 (n = 8), ERK1/2 (n = 7) and PDK1 Ser241(n = 15).
Figure 7.
MYPT1 phosphorylation at Ser668 is increased upon stimulation with the TXA2 analogue, U46619, in permeabilized and intact pulmonary artery SM but not by high [K+] stimulation in intact SM.
(A) U46619 (300 nM) (n = 3) but not high [K+] stimulation increases phosphorylation of MYPT1 Ser668 in intact pulmonary artery smooth muscle. (B) Increasing Ca2+ to pCa7.0 with and without U46619 (300 nM) induced Ca2+ sensitized force in α-toxin permeabilized SM significantly increased phosphorylation of MYPT1 at Ser668 compared to control muscles in G1 solution pCa<8.0 (59±9.6%, n = 10, p<0.01).
Figure 8.
Ribosomal S6 kinase (RSK) signaling scheme for regulation of Ca2+-independendent contraction of smooth muscle.
Agonists through GPCRs leads to activation of sequential ERK1/2 phosphorylations of RSK, subsequent RSK autophosphorylation by its C-terminal kinase domain (CTKD), the recruitment of phosphoinositide-dependent kinase 1 (PDK1) to this newly phosphorylated site, and finally PDK1-dependent phosphorylation at Ser227, with concomitant activation of the NTKD. Active RSK increases RLC20 phosphorylation, either directly or indirectly, as well as inhibitory phosphorylation of MYPT1 at Thr696/853 augmenting Rho/ROCK phosphorylation of these sites. Potential RSK phosphorylation of Ser668 on MYPT1 is also indicated.