Kinase related protein / telokin inhibits Ca2+-independent contraction in triton skinned guinea pig taenia coli
Résumé
The kinase-related protein, KRP, also known as telokin, has been proposed to inhibit smooth muscle contractility by inhibiting the phosphorylation of the regulatory light chains of myosin (rMLC) by the Ca2+-activated myosin light chain kinase (MLCK). Using the phosphatase inhibitor, microcystin, we now show that KRP also inhibits Ca2+-independent rMLC phosphorylation and smooth muscle contraction mediated by novel Ca2+-independent rMLC kinases. Incubating KRP depleted triton skinned taenia coli with microcystin at pCa <8 induced a slow contraction reaching 90% of maximal force (Fmax) at pCa 4.5 after ~25 min. Loading the fibres with KRP significantly slowed down the force development, i.e. the time to reach 50% of Fmax was increased from 8 min to 35 min. KRP similarly inhibited rMLC phosphorylation of heavy meromyosin (HMM) in vitro by MLCK or by the constitutively active MLCK fragment (61K-MLCK) lacking the myosin docking KRP-domain). A C-terminally truncated KRP defective in myosin binding inhibited neither force nor HMM phosphorylation. Phosphorylated KRP inhibited the rMLC phosphorylation of HMM in vitro and Ca2+-insensitive contractions in fibres similar to unphosphorylated KRP, whereby the phosphorylation state of KRP was not altered in the fibres. We conclude that (i) KRP inhibits not only MLCK induced contractions, but also those elicited by Ca2+-independent rMLC kinases; (ii) phosphorylation of KRP does not modulate this effect; (iii) binding of KRP to myosin is essential for this inhibition; and (iv) KRP inhibition of rMLC phosphorylation is most likely due to the shielding of the phosphorylation site on the rMLC.
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