Phosphorylation of the myosin phosphatase targeting subunit and CPI‐17 during Ca2+ Sensitization in Rabbit Smooth Muscle

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 GTPγS concurrently with contraction and myosin phosphorylation in α‐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.