Steady‐state kinetics of skeletal muscle myosin light chain kinase indicate a strong down regulation by products

Geuß, U.; Mayr, Georg W.; Heilmeyer, Ludwig

doi:10.1111/j.1432-1033.1985.tb09305.x

Cited by 28 publications

(17 citation statements)

References 48 publications

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“…For the skeletal isoform interacting with the RLC (47) kinetic studies are consistent with a rapid-equilibrium random bi-bi reaction model. This means that MLCK can bind either RLC or ATP first to form the MLCK · ATP · RLC complex before conversion to the MLCK·ADP·PRLC complex.…”

Section: Mlck Enzyme Activitysupporting

confidence: 53%

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Biochemistry of smooth muscle myosin light chain kinase

Hong

Haldeman

Jackson

et al. 2011

Archives of Biochemistry and Biophysics

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The smooth muscle isoform of myosin light chain kinase (MLCK) is a Ca2+-calmodulin-activated kinase that is found in many tissues. It is particularly important for regulating smooth muscle contraction by phosphorylation of myosin. This review summarizes selected aspects of recent biochemical work on MLCK that pertains to its function in smooth muscle. In general, the focus of the review is on new findings, unresolved issues, and areas with the potential for high physiological significance that need further study. The review includes a concise summary of the structure, substrates, and enzyme activity, followed by a discussion of the factors that may limit the effective activity of MLCK in the muscle. The interactions of each of the many domains of MLCK with the proteins of the contractile apparatus, and the multi-domain interactions of MLCK that may control its behaviors in the cell are summarized. Finally, new in vitro approaches to studying the mechanism of phosphorylation of myosin are introduced.

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Section: Mlck Enzyme Activitysupporting

confidence: 53%

“…The skeletal isoform can form a dead end MLCK·ADP·RLC complex. It is not clear whether this is true for the smooth muscle isoform, which may not be significantly inhibited by ADP (47). This needs to be clarified because free ADP concentrations can be rather high in some smooth muscles.…”

Section: Mlck Enzyme Activitymentioning

confidence: 99%

Biochemistry of smooth muscle myosin light chain kinase

Hong

Haldeman

Jackson

et al. 2011

Archives of Biochemistry and Biophysics

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“…The pattern of inhibition seen with PhK5 and PhK13 is consistent with the phosphorylation reaction mechanism being a sequential rapid-equilibrium random Bi-Bi mechanism, as previously reported (16 -18). The pattern of inhibition seen here is similar to what was reported using synthetic peptides based on the putative autoinhibitory domain of skeletal muscle myosin light chain kinase, an enzyme that also exhibits a rapid-equilibrium random Bi-Bi mechanism (19,20). Those peptides inhibited myosin light chain kinase competitively with respect to phosphate acceptor substrate and noncompetitively with respect to ATP (21).…”

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confidence: 68%

Characterization of the Regulatory Domain of the γ-Subunit of Phosphorylase Kinase

Dasgupta

Blumenthal

1995

Journal of Biological Chemistry

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Phosphorylase kinase is a multimeric protein kinase (␣ 4 ␤ 4 ␥ 4 ␦ 4 ) whose enzymatic activity is conferred by its ␥-subunit. A library of 18 overlapping synthetic peptides spanning residues 277-386 of the ␥-subunit has been prepared to use in identifying important regulatory structures in the protein. In the present study, the library was screened to identify regions that might function as autoinhibitory domains. Peptides from two distinct regions were found to inhibit the Ca 2؉ -activated holoenzyme. The same regions were previously found to bind calmodulin (i.e. the ␦-subunit; Dasgupta, M. Honeycutt, T., and Blumenthal, D. K. (1989) J. Biol. Chem. 264, 17156 -17163). The most potent substrate antagonist peptides were PhK13 (residues 302-326; K i ‫؍‬ 300 nM) and PhK5 (residues 342-366; K i ‫؍‬ 20 M). Both peptides inhibited the holoenzyme competitively with respect to phosphorylase b and noncompetitively with respect to Mg⅐ATP. When the pattern of inhibition with both peptides present was analyzed, inhibition was observed to be synergistic and modestly cooperative indicating that the two peptides can simultaneously occupy the protein substrate-binding site(s). These data are consistent with a model in which the regions of the ␥-subunit represented by PhK5 and PhK13 work in concert as regulatory subdomains that transduce Ca 2؉ -induced conformational changes in the ␦-subunit to the catalytic ␥-subunit through a pseudosubstrate autoinhibitory mechanism.Phosphorylase kinase is among the largest and most complex of the protein kinase superfamily (reviewed by Pickett-Gies and Walsh (1)). Because of its complex structure, the catalytic activity of phosphorylase kinase is sensitive to a variety of effectors and chemical modifications including pH, divalent metal ions, ionic strength and composition, glycogen, calmodulin, nucleosides, phosphorylation state, limited proteolysis, and substrate conformation. The subunit composition of phosphorylase kinase is ␣ 4 ␤ 4 ␥ 4 ␦ 4 , where the ␣-, ␤-, and ␦-subunits are regulatory and the ␥-subunit harbors the catalytic site. The ␣-and ␤-subunits can be phosphorylated, and this modification increases the enzyme's catalytic activity, whereas the ␦-subunit is identical to calmodulin and confers Ca 2ϩ dependence to the enzyme's activity. Although the ␣-, ␤-, and ␦-subunits are all thought to interact directly with the ␥-subunit, the molecular mechanisms by which these regulatory subunits effect changes in catalytic activity is not well understood at present.A common mechanism by which the catalytic activity of many protein kinases appears to be regulated is through a pseudosubstrate autoinhibitory mechanism (see reviews by Soderling (2) and Kemp and Pearson (3)). This intrasteric form of regulation was first described in the cAMP-dependent protein kinase, but there is now substantial evidence that this mechanism may also be operating in the cGMP-dependent protein kinases, myosin light chain kinases, calmodulin-dependent type II kinases, the protein kinase-Cs, as well as the protei...

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“…Under steady state conditions skMLCK follows a rapid-equilibrium random bi-bi reaction (two substrates converted to two products) whereas the smooth muscle MLCK proceeds by an ordered sequential mechanism in which MgATP binds first, followed by the RLC substrate [24, 38, 39]. Stopped-flow, rapid kinetic experiments show activation occurs at a rate indistinguishable from the binding of the Ca 2+ /calmodulin to skMLCK [24].…”

Section: Biochemical Properties Of Mlckmentioning

confidence: 99%

Myosin light chain kinase and the role of myosin light chain phosphorylation in skeletal muscle

Stull

Kamm²,

Vandenboom³

2011

Archives of Biochemistry and Biophysics

156

152

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Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca 2+ /calmodulin-dependent serine-threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca 2+ binding to calmodulin forming a (Ca 2+ ) 4 •calmodulin complex sufficient for activation with a diffusion limited, stoichiometic binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca 2+ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca 2+ / calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca 2+ -sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue.

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Steady‐state kinetics of skeletal muscle myosin light chain kinase indicate a strong down regulation by products

Cited by 28 publications

References 48 publications

Biochemistry of smooth muscle myosin light chain kinase

Biochemistry of smooth muscle myosin light chain kinase

Characterization of the Regulatory Domain of the γ-Subunit of Phosphorylase Kinase

Myosin light chain kinase and the role of myosin light chain phosphorylation in skeletal muscle

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