title: Kinase-related protein/telokin inhibits Ca2+-independent contraction in Triton-skinned guinea pig taenia coli.
abstract: KRP (kinase-related protein), also known as telokin, has been proposed to inhibit smooth muscle contractility by inhibiting the phosphorylation of the rMLC (regulatory myosin light chain) by the Ca2+-activated MLCK (myosin light chain kinase). Using the phosphatase inhibitor microcystin, we show in the present study 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 approximately 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 HMM (heavy meromyosin) 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 probably due to the shielding of the phosphorylation site on the rMLC.
authors.affiliation: Laboratory of Cell Motility, Institute of Experimental Cardiology at the Russian Cardiology and Production Research Centre, 3rd Cherepkovskaya Str. 15a, Moscow 121552, Russia.
authors.forename: Olga V
authors.initials: OV
authors.lastname: Shcherbakova
authors.forename: Daria V
authors.initials: DV
authors.lastname: Serebryanaya
authors.forename: Alexander B
authors.initials: AB
authors.lastname: Postnikov
authors.forename: Mechthild M
authors.initials: MM
authors.lastname: Schroeter
authors.forename: Stefan
authors.initials: S
authors.lastname: Zittrich
authors.forename: Angelika A
authors.initials: AA
authors.lastname: Noegel
authors.forename: Vladimir P
authors.initials: VP
authors.lastname: Shirinsky
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Vorotnikov
authors.forename: Gabriele
authors.initials: G
authors.lastname: Pfitzer
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D002645:Chickens; D003106:Colon / Q000187:drug effects / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D005260:Female; D006168:Guinea Pigs; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008387:Marine Toxins; D052998:Microcystins / Q000494:pharmacology; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008954:Models, Biological; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D009130:Muscle, Smooth / Q000187:drug effects / Q000502:physiology; D018994:Myosin Light Chains / Q000737:chemistry / Q000378:metabolism; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D017830:Octoxynol; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
chemical_list: D002135:Calcium-Binding Proteins; D017931:DNA Primers; D008387:Marine Toxins; D052998:Microcystins; D018994:Myosin Light Chains; D015879:Myosin Subfragments; D010446:Peptide Fragments; D011994:Recombinant Proteins; C060640:telokin; D017830:Octoxynol; D017868:Cyclic AMP-Dependent Protein Kinases; C499378:MYLK protein, human; D009219:Myosin-Light-Chain Kinase; D048052:Mitogen-Activated Protein Kinase 3; C057862:cyanoginosin LR; D002118:Calcium