A temporal Ca2+-desensitization of myosin light chain kinase in phasic smooth muscles induced by CaMKKß/PP2A pathways.

Kitazawa T, Matsui T, Katsuki S, Goto A, Akagi K, Hatano N, Tokumitsu H, Takeya K, Eto M.

Cell signaling pathways regulating myosin regulatory light chain (LC20) phosphorylation contribute to determining contractile responses in smooth muscles. Following excitation and contraction, phasic smooth muscles, such as digestive tract and urinary bladder, undergo a relaxation due to a decline of cellular [Ca2+] and a decreased Ca2+ sensitivity of LC20 phosphorylation, named Ca2+ desensitization. Here, we determined mechanisms underlying the temporal Ca2+ desensitization of LC20 phosphorylation in phasic smooth muscles using permeabilized strips of mouse ileum and urinary bladder. Upon the stimulation with pCa6.0 at 20°C, the contraction and the LC20 phosphorylation peaked within 30 sec and then declined to about 50% of the peak force at 2 min after stimulation. During the relaxation phase after the contraction, the LC20 kinase (MLCK) was inactivated, but no fluctuation in the LC20 phosphatase activity occurred, suggesting that the MLCK inactivation is a cause of the Ca2+-induced Ca2+-desensitization of LC20 phosphorylation. The MLCK inactivation was associated with phosphorylation at the calmodulin binding domain of the kinase. Treatment with antagonists for CaMKKß (STO-609 and TIM-063) attenuated both the phasic response of the contraction and MLCK phosphorylation, whereas neither CaMKII, AMPK nor PAK induced the MLCK inactivation in phasic smooth muscles. Conversely, PP2A inhibition amplified the phasic response. Signaling pathways through CaMKKß and PP2A may contribute to regulating the Ca2+ sensitivity of MLCK and the contractile response of phasic smooth muscles.

American Journal of Physiology-Cell Physiology

321

3

C549

C558