Phosphorylation of the myosin phosphatase targeting subunit and CPI-17 during Ca2+ sensitization in rabbit smooth muscle

Abstract

Myosin phosphatase (MLCP) plays a critical regulatory role in the Ca(2+) sensitivity of myosin phosphorylation and smooth muscle contraction. It has been suggested that phosphorylation at Thr(695) of the MLCP regulatory subunit (MYPT1) and at Thr(38) of the MLCP inhibitor protein CPI-17 results in inhibition of MLCP activity. We have previously demonstrated that CPI-17 Thr(38) phosphorylation plays an important role in G-protein-mediated inhibition of MLCP in tonic arterial smooth muscle. Here, we attempted to evaluate the function of MYPT1 in phasic rabbit portal vein (PV) and vas deferens (VD) smooth muscles. Using site- and phospho-specific antibodies, phosphorylation of MYPT1 Thr(695) and CPI-17 Thr(38) was examined along with MYPT1 Thr(850), which is a non-inhibitory Rho-kinase site. We found that both CPI-17 Thr(38) and MYPT1 Thr(850) were phosphorylated in response to agonists or GTPgammaS concurrently with contraction and myosin phosphorylation in alpha-toxin-permeabilized PV tissues. In contrast, phosphorylation of MYPT1 Thr(695) did not increase. Comparable results were also obtained in both permeabilized and intact VD. The Rho-kinase inhibitor Y-27632 and the protein kinase C (PKC) inhibitor GF109203X suppressed phosphorylation of MYPT1 Thr(850) and CPI-17 Thr(38), respectively, in intact VD while MYPT1 Thr(695) phosphorylation was insensitive to both inhibitors. These results indicate that phosphorylation of MYPT1 Thr(695) is independent of stimulation of G-proteins, Rho-kinase or PKC. In the phasic PV, phosphorylation of CPI-17 Thr(38) may contribute towards inhibition of MLCP while the phasic visceral VD, which has a low CPI-17 concentration, probably utilizes other Ca(2+) sensitizing mechanisms for inhibiting MLCP besides phosphorylation of MYPT1 and CPI-17.

Figure 1. G-protein-mediated Ca²⁺ sensitization of contraction and myosin phosphorylation in rabbit portal vein (PV) smooth muscle

Effects of rigor, pCa > 8, pCa 6.3 alone, pCa 6.3 + phenylephrine (PE) and pCa 6.3 + GTPγS on contraction (A) and MLC phosphorylation (B) in α-toxin-permeabilized rabbit portal vein (PV) smooth muscle. Rigor: after permeabilized with α-toxin at pCa 6.3 and treated with A23187 in the Ca²⁺-free relaxing solution, the PV strips were incubated in the Ca²⁺-free, MgATP-free, creatine phosphate-free solution for 30 min at 20 °C and then frozen. pCa > 8: the permeabilized strips were soaked in the 10 mm EGTA-containing relaxing solution for 30 min. pCa 6.3: after incubation in the relaxing solution for 30 min, the strips were stimulated with the pCa 6.3-containing solution for 13 min. pCa 6.3 + PE: the strips were first incubated in the pCa 6.3 solution alone for 10 min and then stimulated with the same solution except containing 30 µm PE for 3 min. pCa 6.3 + GTPγS: the strips were treated with the same procedure as in the pCa 6.3 + PE except that GTPγS was added instead of PE during stimulation. Force levels were normalized with the maximal levels developed at pCa 4.5 plus 30 µm GTPγS in individual strips. Phosphorylation levels of MLC were measured as described in Methods. The number of experiments was 8–13.

Figure 2. Phosphorylation of CPI-17 and MYPT1 in rabbit portal vein

The figure shows in vitro phosphorylation of recombinant MYPT1 by Rho-kinase (A) and in vivo phosphorylation of CPI-17 Thr³⁸ (B), MYPT1 Thr⁸⁵⁰ (C), and MYPT1 Thr⁶⁹⁵ (D) at rigor, pCa 6.3 alone, pCa 6.3 + PE and pCa 6.3 + GTPγS in α-toxin-permeabilized rabbit PV smooth muscle. A, phosphorylation of GST-MYPT1 (654–880) with [γ-³²P]ATP was performed as described in Methods. Upper panels show immunoblots using anti-p[Thr⁶⁹⁵] (left) and anti-p[Thr⁸⁵⁰] (right). Lower panels show their autoradiograms. In B, C and D, upper panel shows a representative paired set of Western blots of total and phosphorylated CPI-17 Thr³⁸, MYPT1 Thr⁸⁵⁰ and MYPT1 Thr⁶⁹⁵, respectively. Average phosphorylation levels (lower panel) of CPI-17 Thr³⁸ (B), MYPT1 Thr⁸⁵⁰ (C) and MYPT1 Thr⁶⁹⁵ (D) are expressed as a percentage of a value induced by 30 µm GTPγS at pCa 6.3 for each site. The number of experiments was 3–4 for B, C and D.

Figure 3. Uncoupling of GTPγS-induced force generation and phosphorylation of MYPT1 at Thr⁶⁹⁵ in rabbit vas deferens (VD)

The figure shows effects of rigor, pCa > 8, pCa 6.3 alone and pCa 6.3 + GTPγS on contraction (A), CPI-17 Thr³⁸ phosphorylation (B), MYPT1 Thr⁸⁵⁰ phosphorylation (C), and MYPT1 Thr⁶⁹⁵ phosphorylation (D) in α-toxin-permeabilized rabbit vas deferens (VD) smooth muscle. Conditions used were the same as those in Figs 1 and 2, except that different smooth muscle tissues were used (n = 3–6).

Figure 4. Uncoupling of agonist-induced force generation and phosphorylation of MYPT1 Thr⁶⁹⁵ in intact rabbit VD

The figure shows contraction (A), CPI-17 Thr³⁸ phosphorylation (B), MYPT1 Thr⁸⁵⁰ phosphorylation (C), and MYPT1 Thr⁶⁹⁵ phosphorylation (D) at rest or during contraction stimulated with either high K⁺, PE, or endothelin-1 (ET) in intact rabbit VD smooth muscle. Force and phosphorylation levels were measured in the normal external solution either at rest, 15 s after high K⁺ was applied (K-peak), 10 min after high K⁺ (K-plateau), 1.5 min after 30 µm PE or 3 min after 1 µm ET. Force levels were normalized to the peak of high K⁺-induced contraction. The relative phosphorylation was determined as described in Methods. The mean phosphorylation at rest was expressed as 1 (n = 4–7).

Figure 5. Rho-kinase independent phosphorylation of MYPT1 Thr⁶⁹⁵ in intact VD

Effect of 1 µm prazosin, 10 µm Y-27632 or 3 µm GF109203X on contraction (A), CPI-17 Thr³⁸ phosphorylation (B), MYPT1 Thr⁸⁵⁰ phosphorylation (C), and MYPT1 Thr⁶⁹⁵ phosphorylation (D) is shown either at rest, at the peak (high K⁺ peak) or during the plateau phase of high K⁺-induced contraction (high K⁺ plateau) in intact rabbit VD smooth muscle. The inhibitors were applied from 10 min before stimulations throughout experiments. The strips were stimulated and frozen as described in Fig. 3 (n = 4–7).