Possible roles of N- and C-terminal unstructured tails of CPI-17 in regulating Ca2+ sensitization force of smooth muscle.

CPI-17 regulates the myosin phosphatase and mediates the agonist-induced contraction of smooth muscle. PKC and ROCK phosphorylate CPI-17 at Thr38 leading to a conformational change of the central inhibitory domain (PHIN domain). The N- and C-terminal tails of CPI-17 are predicted as unstructured loops and their sequences are conserved among mammals. Here we characterized CPI-17 N- and C-terminal unstructured tails using recombinant proteins that lack the potions. Recombinant CPI-17 proteins at a physiologic level (10 µM) were doped into beta-escin-permeabilized smooth muscle strips for Ca2+ sensitization force measurement. The ectopic full-length CPI-17 augmented the PDBu-induced Ca2+ sensitization force at pCa6.3, indicating myosin phosphatase inhibition. Deletion of N- and C-terminal tails of CPI-17 attenuated the extent of PDBu-induced Ca2+-sensitization force. The N-terminal deletion dampened phosphorylation at Thr38 by protein kinase C (PKC), and the C-terminal truncation lowered the affinity to the myosin phosphatase. Under the physiologic conditions, PKC and myosin phosphatase may recognize CPI-17 N-/C-terminal unstructured tails inducing Ca2+ sensitization force in smooth muscle cells.

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

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 the digestive tract and urinary bladder, undergo relaxation due to a decline of cellular Ca2+ concentration and decreased Ca2+ sensitivity of LC20 phosphorylation, named Ca2+ desensitization. Here, we determined the mechanisms underlying the temporal Ca2+ desensitization of LC20 phosphorylation in phasic smooth muscles using permeabilized strips of the mouse ileum and urinary bladder. Upon stimulation with pCa6.0 at 20°C, contraction and LC20 phosphorylation peaked within 30 s and then declined to about 50% of the peak force at 2 min after stimulation. During the relaxation phase after the contraction, LC20 kinase [myosin light chain kinase (MLCK)] was inactivated, but no fluctuation in LC20 phosphatase activity occurred, suggesting that MLCK inactivation is a cause of the Ca2+-induced Ca2+ desensitization of LC20 phosphorylation. MLCK inactivation was associated with phosphorylation at the calmodulin-binding domain of the kinase. Treatment with STO-609 and TIM-063 antagonists for Ca2+/calmodulin (CaM)-dependent protein kinase kinase-ß (CaMKKß) attenuated both the phasic response of the contraction and MLCK phosphorylation, whereas neither CaM kinase II, AMP-activated protein kinase, nor p21-activated kinase induced MLCK inactivation in phasic smooth muscles. Conversely, protein phosphatase 2A inhibition amplified the phasic response. Signaling pathways through CaMKKß and protein phosphatase 2A may contribute to regulating the phasic response of smooth muscle contraction.

Kinase activity-tagged western blotting assay.

Determining cellular activities of protein kinases is a fundamental step for characterizing pathophysiological cell signaling pathways. Here, we optimized a nonradioactive method that detects protein kinases in tissues or cells after separation by SDS-PAGE and transfer onto polyvinylidene fluoride membranes. The method, kinase activity-tagged western blotting (KAT-WB), consists of five steps: electrophoresis of cell extracts that contain protein kinases, electroblotting proteins onto polyvinylidene fluoride membrane, denaturation-renaturation, phosphorylation, with or without an added substrate protein and immunodetection using anti-phospho-specific antibodies. KAT-WB detected autophosphorylation of one Tyr-kinase and site-specific phosphorylation of added substrate by multiple kinases. KAT-WB assay enables us to interrogate multiple kinase signaling pathways without using radioactive ATP.