Effects of myosin kinase inhibiting peptide on contractility and LCzO phosphorylation in skinned smooth muscle JOHN
D. STRAUSS,
PRIMAL
DE LANEROLLE,
AND RICHARD
J. PAUL
Departments of Physiology and Biophysics and of Pharmacology and Cell Biophysics, University of Cincinnati, Cincinnati, Ohio 45267-0576; and Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612 Strauss, John D., Primal de Lanerolle, and Richard J. Paul. Effects of myosin kinase inhibiting peptide on contractility and LC,,, phosphorylation in skinned smooth muscle. Am. J. Physiol. 262 (CelZ Physiol. 31): C1437-C1445, 1992.-A peptide inhibitor, myosin kinase inhibitor (MKI), of myosin light chain kinase (MLCK) was tested for its effects on contractility and myosin light chain phosphorylation in Triton X-100 skinned guinea pig taenia coli. MKI is based on the amino acid sequence of the myosin light chain (residues 11-19 LC,,) and is a competitive inhibitor [inhibitory constant (K;) N 10 PM] of purified MLCK with respect to myosin light chains (LC,,). MKI inhibited unloaded shortening velocity (Vu,) and the calcium-sensitive ATPase activity of the skinned fibers but had no significant effect on steady-state isometric force or myosin light chain phosphorylation, as measured by IEF-polyacrylamide gel electrophoresis analysis. MKI had no significant effect on V,,, of thiophosphorylated fibers in the absence of calcium. MKI inhibited MLCK activity in protein extracts from taenia coli, as measured by radioactive phosphate incorMKI also inhibited the phosporation into LC,,. Surprisingly, phatase activity of these same extracts. This peptide slowed the rate and extent of relaxation of calcium-contracted fibers and elicited a contraction in relaxed fibers. These results are consistent with the hypothesis that MKI may be a phosphatase inhibitor as well as an inhibitor of MLCK. Our data further suggest that the rate of phosphorylation-dephosphorylation turnover may be important in regulating Vu, in smooth muscle. smooth muscle regulation; myosin phosphorylation; peptide inhibitors; phosphatase inhibition IS WELL KNOWN that many smooth muscles develop force monotonically and maintain tension in response to a sustained stimulus. There is substantial evidence demonstrating that the Ca2+-dependent phosphorylation of the ZO-kDa light chains of myosin (LC,,) by myosin light chain kinase (MLCK) is essential for the initiation of this contraction (for reviews, see Refs. 8, 16). However, changes in myosin light chain phosphorylation (MLC-Pi) 9 ATP utilization, intracellular Ca2+, and unloaded shortening velocity (Vu,) follow biphasic time courses in response to a sustained stimulus. The condition of tension maintenance with reduced shortening velocity has been denoted by Murphy and colleagues as the latch state (for review, see Ref. 15). A number of hypotheses have been proposed for regulation of the latch state including thin filament-linked systems involving leiotonin, caldesmon, and calponin, direct binding of Ca2+ to myosin, alternate site phosphorylation by MLCK and other kinases, and dephosphorylation of attached cross bridges (8, 16). It has not yet been established which of these mechanisms, if any, have a physiological role in regulating smooth muscle contractility. One strategy to better define the role of LC20 phosphorylation is selective inhibition of MLC-Pi through
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$2.00
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specific inhibition of MLCK. For example, calmodulin antagonists have been used to remove the influence of MLCK by affecting the binding of the activating Ca2+calmodulin complex; these include the phenothiazines (6) and RS20, a synthetic peptide based on the amino acid sequence of MLCK (29). Other modulators of MLCK activity, including ML9, &chloronaphthalenesulfonyl-lH-hexahydro-1,4,diazepine (18), and H8, an isoquinolinesulfonamide (14)) have been tested for their effects on smooth muscle contractility. Each of these was intended to reduce the extent of LC20 phosphorylation in the presence of activating levels of Ca2+. However, these agents are subject to questions concerning the specificity of action with respect to inhibiting MLCK activity, and other agents may be necessary to test these hypotheses (2, 16). Recently, interest has grown in using synthetic peptides as probes for specific kinases. Synthetic peptides may be designed to take advantage of an enzyme’s binding specificities based upon knowledge of its structure and substrate. Thus specifically targeted competitive inhibitors have been synthesized and are valuable probes in the study of enzyme biochemistry (19). Myosin kinase inhibitor (MKI), a nine-amino acid peptide based on the amino acid sequence around the phosphorylation site of L&o, is a synthetic peptide specifically designed to inhibit MLCK (26). It inhibits the in vitro activity of MLCK with an apparent inhibitory constant (Ki) of 10 PM (17, 26) which is in the same range as the K, of MLCK for the native light chain (see Ref. 19 for review). We have used chemically demembranated (skinned) smooth muscle to investigate the effects of this peptide on contractility. Skinned smooth muscle retains contractile function while being permeable to large bioactive molecules (see Ref. 20 for review) such as the catalytic subunit of adenosine 3’,5’-cyclic monophosphatedependent protein kinase, MLCK, myosin phosphatases (8, 28), and antibodies against MLCK (9). Moreover, a peptide inhibitor of calmodulin has been used to modulate contractile properties of skinned smooth muscle (29). Data from experiments performed with MKI and skinned taenia coli support the hypothesis that the turnover rate of protein phosphorylation may be important in regulating the contractile properties of smooth muscle. METHODS
Skinned fibers. Taenia coli were dissected from guinea pig and skinned as described previously (23). Briefly, strips of tissue were calcium depleted in a high-potassium ethylene glycolhis@-aminoethyl ether)-N,N,N’,iV’-tetraacetic acid (EGTA)buffered solution, pH 7.0. This was followed by treatment with
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpcell at Lunds Univ Medicinska Fak Biblio (130.235.066.010) on February 14, 2019.
Cl437
Cl438
EFFECTS
OF MKI
IN
SKINNED
a similar solution containing 1% Triton X-100 for 4 h to solubilize cell membranes. The fiber bundles were stored at -30°C in a solution containing 50% glycerol (vol/vol), 4 mM EGTA, 1 mM NaN,, 7.5 mM ATP, 10 mM MgCl,, and ‘20 mM imidazole, pH 6.7, at least overnight and up to 1 mo before use. Solutions. MKI (Lys-Lys-Arg-Ala-Ala-Arg-Ala-Thr-SerNH2) (17, 26) was obtained primarily from Peninsula Laboratories. Amino acid composition and sequence were verified on site using the Protein Chemistry core facility of the Department of Pharmacology and Cell Biophysics, University of Cincinnati, under the supervision of Dr. Terence Kirley. Additional quantities of peptide were provided by Dr. Suzanne Moreland of the Squibb Institute for Medical Research, as an independent source, to verify results using the batches of Peninsula Laboratories peptide. All other chemicals, except as specifically noted, were obtained from Sigma. Relaxing solution consisted of 10 mM MgC12, 1 mM NaN3, 7.5 mM Na, ATP, 4 mM EGTA, 0.1 PM calmodulin (from either Sigma Chemical or Boehringer Mannheim), 20 mM imidazole (pH 6.7), 10 mM phosphocreatine, and 10 U/ml creatine kinase. Contracting solution was similar to relaxing solution but also contained 2.0 mM CaCl,, resulting in a buffered free Ca2+ concentration of -1.54 PM, 1.94 mM free Mg2+, 7.2 mM Mg ATP, and an ionic strength of 110 mM, based on calculations from computer programs adapted for IBM personal computers from those used by Godt and Maughan (12). Relaxing solution contained Ca2+ at
D. STRAUSS,
PRIMAL
DE LANEROLLE,
AND RICHARD
J. PAUL
Departments of Physiology and Biophysics and of Pharmacology and Cell Biophysics, University of Cincinnati, Cincinnati, Ohio 45267-0576; and Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612 Strauss, John D., Primal de Lanerolle, and Richard J. Paul. Effects of myosin kinase inhibiting peptide on contractility and LC,,, phosphorylation in skinned smooth muscle. Am. J. Physiol. 262 (CelZ Physiol. 31): C1437-C1445, 1992.-A peptide inhibitor, myosin kinase inhibitor (MKI), of myosin light chain kinase (MLCK) was tested for its effects on contractility and myosin light chain phosphorylation in Triton X-100 skinned guinea pig taenia coli. MKI is based on the amino acid sequence of the myosin light chain (residues 11-19 LC,,) and is a competitive inhibitor [inhibitory constant (K;) N 10 PM] of purified MLCK with respect to myosin light chains (LC,,). MKI inhibited unloaded shortening velocity (Vu,) and the calcium-sensitive ATPase activity of the skinned fibers but had no significant effect on steady-state isometric force or myosin light chain phosphorylation, as measured by IEF-polyacrylamide gel electrophoresis analysis. MKI had no significant effect on V,,, of thiophosphorylated fibers in the absence of calcium. MKI inhibited MLCK activity in protein extracts from taenia coli, as measured by radioactive phosphate incorMKI also inhibited the phosporation into LC,,. Surprisingly, phatase activity of these same extracts. This peptide slowed the rate and extent of relaxation of calcium-contracted fibers and elicited a contraction in relaxed fibers. These results are consistent with the hypothesis that MKI may be a phosphatase inhibitor as well as an inhibitor of MLCK. Our data further suggest that the rate of phosphorylation-dephosphorylation turnover may be important in regulating Vu, in smooth muscle. smooth muscle regulation; myosin phosphorylation; peptide inhibitors; phosphatase inhibition IS WELL KNOWN that many smooth muscles develop force monotonically and maintain tension in response to a sustained stimulus. There is substantial evidence demonstrating that the Ca2+-dependent phosphorylation of the ZO-kDa light chains of myosin (LC,,) by myosin light chain kinase (MLCK) is essential for the initiation of this contraction (for reviews, see Refs. 8, 16). However, changes in myosin light chain phosphorylation (MLC-Pi) 9 ATP utilization, intracellular Ca2+, and unloaded shortening velocity (Vu,) follow biphasic time courses in response to a sustained stimulus. The condition of tension maintenance with reduced shortening velocity has been denoted by Murphy and colleagues as the latch state (for review, see Ref. 15). A number of hypotheses have been proposed for regulation of the latch state including thin filament-linked systems involving leiotonin, caldesmon, and calponin, direct binding of Ca2+ to myosin, alternate site phosphorylation by MLCK and other kinases, and dephosphorylation of attached cross bridges (8, 16). It has not yet been established which of these mechanisms, if any, have a physiological role in regulating smooth muscle contractility. One strategy to better define the role of LC20 phosphorylation is selective inhibition of MLC-Pi through
IT
0363-6143/92
$2.00
Copyright
specific inhibition of MLCK. For example, calmodulin antagonists have been used to remove the influence of MLCK by affecting the binding of the activating Ca2+calmodulin complex; these include the phenothiazines (6) and RS20, a synthetic peptide based on the amino acid sequence of MLCK (29). Other modulators of MLCK activity, including ML9, &chloronaphthalenesulfonyl-lH-hexahydro-1,4,diazepine (18), and H8, an isoquinolinesulfonamide (14)) have been tested for their effects on smooth muscle contractility. Each of these was intended to reduce the extent of LC20 phosphorylation in the presence of activating levels of Ca2+. However, these agents are subject to questions concerning the specificity of action with respect to inhibiting MLCK activity, and other agents may be necessary to test these hypotheses (2, 16). Recently, interest has grown in using synthetic peptides as probes for specific kinases. Synthetic peptides may be designed to take advantage of an enzyme’s binding specificities based upon knowledge of its structure and substrate. Thus specifically targeted competitive inhibitors have been synthesized and are valuable probes in the study of enzyme biochemistry (19). Myosin kinase inhibitor (MKI), a nine-amino acid peptide based on the amino acid sequence around the phosphorylation site of L&o, is a synthetic peptide specifically designed to inhibit MLCK (26). It inhibits the in vitro activity of MLCK with an apparent inhibitory constant (Ki) of 10 PM (17, 26) which is in the same range as the K, of MLCK for the native light chain (see Ref. 19 for review). We have used chemically demembranated (skinned) smooth muscle to investigate the effects of this peptide on contractility. Skinned smooth muscle retains contractile function while being permeable to large bioactive molecules (see Ref. 20 for review) such as the catalytic subunit of adenosine 3’,5’-cyclic monophosphatedependent protein kinase, MLCK, myosin phosphatases (8, 28), and antibodies against MLCK (9). Moreover, a peptide inhibitor of calmodulin has been used to modulate contractile properties of skinned smooth muscle (29). Data from experiments performed with MKI and skinned taenia coli support the hypothesis that the turnover rate of protein phosphorylation may be important in regulating the contractile properties of smooth muscle. METHODS
Skinned fibers. Taenia coli were dissected from guinea pig and skinned as described previously (23). Briefly, strips of tissue were calcium depleted in a high-potassium ethylene glycolhis@-aminoethyl ether)-N,N,N’,iV’-tetraacetic acid (EGTA)buffered solution, pH 7.0. This was followed by treatment with
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpcell at Lunds Univ Medicinska Fak Biblio (130.235.066.010) on February 14, 2019.
Cl437
Cl438
EFFECTS
OF MKI
IN
SKINNED
a similar solution containing 1% Triton X-100 for 4 h to solubilize cell membranes. The fiber bundles were stored at -30°C in a solution containing 50% glycerol (vol/vol), 4 mM EGTA, 1 mM NaN,, 7.5 mM ATP, 10 mM MgCl,, and ‘20 mM imidazole, pH 6.7, at least overnight and up to 1 mo before use. Solutions. MKI (Lys-Lys-Arg-Ala-Ala-Arg-Ala-Thr-SerNH2) (17, 26) was obtained primarily from Peninsula Laboratories. Amino acid composition and sequence were verified on site using the Protein Chemistry core facility of the Department of Pharmacology and Cell Biophysics, University of Cincinnati, under the supervision of Dr. Terence Kirley. Additional quantities of peptide were provided by Dr. Suzanne Moreland of the Squibb Institute for Medical Research, as an independent source, to verify results using the batches of Peninsula Laboratories peptide. All other chemicals, except as specifically noted, were obtained from Sigma. Relaxing solution consisted of 10 mM MgC12, 1 mM NaN3, 7.5 mM Na, ATP, 4 mM EGTA, 0.1 PM calmodulin (from either Sigma Chemical or Boehringer Mannheim), 20 mM imidazole (pH 6.7), 10 mM phosphocreatine, and 10 U/ml creatine kinase. Contracting solution was similar to relaxing solution but also contained 2.0 mM CaCl,, resulting in a buffered free Ca2+ concentration of -1.54 PM, 1.94 mM free Mg2+, 7.2 mM Mg ATP, and an ionic strength of 110 mM, based on calculations from computer programs adapted for IBM personal computers from those used by Godt and Maughan (12). Relaxing solution contained Ca2+ at
