Phosphorylation of caldesmon by ERK MAP kinases in smooth muscle

Phosphorylation of h-caldesmon has beenproposed to regulate airway smooth muscle contraction. Bothextracellular signal-regulated kinase (ERK) and p38 mitogen-activatedprotein (MAP) kinases phosphorylate h-caldesmon in vitro. To determinewhether both enzymes phosphorylate caldesmon in vivo,phosphorylation-site-selective antibodies were used to assayphosphorylation of MAP kinase consensus sites. Stimulation of culturedtracheal smooth muscle cells with ACh or platelet-derived growth factorincreased caldesmon phosphorylation at Ser789 by about twofold.Inhibiting ERK MAP kinase activation with 50 µM PD-98059 blockedagonist-induced caldesmon phosphorylation completely. Inhibiting p38MAP kinases with 25 µM SB-203580 had no effect on ACh-inducedcaldesmon phosphorylation. Carbachol stimulation increased caldesmonphosphorylation at Ser789 in intact tracheal smooth muscle, which wasblocked by the M₂ antagonist AF-DX 116 (1 µM). AF-DX 116 inhibited carbachol-induced isometric contraction by 15 ± 1.4%, thusdissociating caldesmon phosphorylation from contraction. Activation ofM₂ receptors leads to activation of ERK MAP kinases andphosphorylation of caldesmon with little or no functional effect onisometric force. P38 MAP kinases are also activated by muscarinicagonists, but they do not phosphorylate caldesmon in vivo.