Role of basic residues in the phosphorylation of synthetic peptides by myosin light chain kinase.

Kemp, Bruce E.; Pearson, Richard B.; House, Colin M.

doi:10.1073/pnas.80.24.7471

Cited by 80 publications

(39 citation statements)

References 22 publications

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“…Thus, among the substrates tested the kinase shows a high selectivity for the KM-14 sequence, and alterations in the amino terminal sequence of the peptide reduces its suitability as a substrate. This is consistent with previous observations with smooth muscle MLCK (14).…”

Section: Resultssupporting

confidence: 83%

“…31). MLCK has a very narrow specificity, and it is clear that the content and orientation of the arginine and lysine residues in the KM-14 sequence are critical for optimal catalytic activity (14). In contrast, the multifunctional calmodulin-dependent protein kinase has a less stringent requirement for substrate recognition (Arg-x-xSer, with x representing any amino acid).…”

Section: Resultsmentioning

confidence: 99%

“…As shown in Table IV (12) and rabbit myosin light chains (2) by Mougeotia extracts, the KM-14 kinase from Mougeotia was selected for further study. The enzyme (20-27 ,uM) reported for the same peptide with chicken gizzard myosin light chain kinase (14,27).…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…The sequence of this substrate is based on the site on the regulatory light chain of chicken gizzard myosin that is necessary for recognition and phosphorylation by calmodulin-dependent MLCK (14,27). Phosphorylation occurs on serine 9 of the peptide (14). As shown in Table I, in the absence of added substrate, phosphorylation of endogenous proteins was not detected under the conditions of the filter paper assay (see "Materials and Methods" for details of this assay).…”

Section: Resultsmentioning

confidence: 99%

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Detection of a Calcium-Activated Protein Kinase in Mougeotia by Using Synthetic Peptide Substrates

Roberts

1989

Plant Physiol.

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By using a synthetic peptide, KM-14, a protein kinase was detected and partially purified from Mougeotia sp. The peptide contains the sequence of the regulatory light chain of smooth muscle myosin that is phosphorylated by calcium-calmodulindependent myosin light chain kinase (MLCK). The Mougeotia kinase was stimulated 40-fold by calcium with half-maximal stimulation occurring at 1.5 micromolar. The enzyme was fractionated from calmodulin and was depleted of calmodulin based on enzyme activator analysis. The calmodulin-depleted enzyme was fully active and calcium dependent, and was not stimulated further by exogenous calmodulin nor by the calcium effectors phosphatidylserine and diacylglycerol. The enzyme phosphorylated intact chicken gizzard myosin light chain as well as the KM-14 substrate. KM-13, a peptide analog of KM-14 with a deletion of a glutamine at position 5, was a poor substrate with a V.I/Km ratio 200-fold lower than KM-14. Thus, similarly to vertebrate MLCK, the Mougeotia enzyme is very sensitive to changes in sequence surrounding the phosphorylation site. Calcium-dependent KM-14 kinase activity also was detected in two other algae, Mesotaenium caldariorum and Spirogyra sp., as well as in pea seedlings. The data suggest that plant and algal tissues possess an enzyme with a substrate specificity similar to MLCK, but unlike MLCK, does not appear to require calmodulin for activity.

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Section: Resultssupporting

confidence: 83%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 3 more Smart Citations

Detection of a Calcium-Activated Protein Kinase in Mougeotia by Using Synthetic Peptide Substrates

Roberts

1989

Plant Physiol.

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Myosin light chain kinase steady‐state kinetics: comparison of smooth muscle myosin II and nonmuscle myosin IIB as substrates

Alcala

Haldeman

Brizendine

et al. 2016

Cell Biochemistry & Function

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Myosin light chain kinase (MLCK) phosphorylates S19 of the myosin regulatory light chain (RLC), which is required to activate myosin's ATPase activity and contraction. Smooth muscles are known to display plasticity in response to factors such as inflammation, developmental stage, or stress, which lead to differential expression of nonmuscle and smooth muscle isoforms. Here, we compare steady-state kinetics parameters for phosphorylation of different MLCK substrates: (1) nonmuscle RLC, (2) smooth muscle RLC, and heavy meromyosin subfragments of (3) nonmuscle myosin IIB, and (4) smooth muscle myosin II. We show that MLCK has a ~2-fold higher kcat for both smooth muscle myosin II substrates compared with nonmuscle myosin IIB substrates, whereas Km values were very similar. Myosin light chain kinase has a 1.6-fold and 1.5-fold higher specificity (kcat/Km) for smooth versus nonmuscle-free RLC and heavy meromyosin, respectively, suggesting that differences in specificity are dictated by RLC sequences. Of the 10 non-identical RLC residues, we ruled out 7 as possible underlying causes of different MLCK kinetics. The remaining 3 residues were found to be surface exposed in the N-terminal half of the RLC, consistent with their importance in substrate recognition. These data are consistent with prior deletion/chimera studies and significantly add to understanding of MLCK myosin interactions.

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Vascular smooth muscle myosin light chain diphosphorylation: Mechanism, function, and pathological implications

Walsh

2011

IUBMB Life

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SummarySmooth muscle contraction is activated primarily by phosphorylation at S19 of the 20-kDa regulatory light chain subunits of myosin II (LC 20 ) catalyzed by Ca 21 /calmodulin-dependent myosin light chain kinase. Other kinases, for example, integrinlinked kinase (ILK), Rho-associated kinase (ROCK), and zipper-interacting protein kinase (ZIPK), can phosphorylate T18 in addition to S19, which increases the actin-activated myosin MgATPase activity at subsaturating actin concentrations 3-fold. These phosphorylatable residues and the amino acid sequence surrounding them are highly conserved throughout the animal kingdom; they are also found in an LC 20 homolog within the genome of Monosiga brevicollis, the closest living relative of metazoans. LC 20 diphosphorylation has been detected in mammalian vascular smooth muscle tissues in response to specific contractile stimuli and in pathophysiological situations associated with hypercontractility. LC 20 diphosphorylation has also been observed frequently in cultured cells where it activates force generation. Kinases such as ILK, ROCK, and ZIPK, therefore, are potential therapeutic targets in the treatment of, for example, cerebral vasospasm following subarachnoid hemorrhage and atherosclerosis.

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Role of basic residues in the phosphorylation of synthetic peptides by myosin light chain kinase.

Cited by 80 publications

References 22 publications

Detection of a Calcium-Activated Protein Kinase in Mougeotia by Using Synthetic Peptide Substrates

Detection of a Calcium-Activated Protein Kinase in Mougeotia by Using Synthetic Peptide Substrates

Myosin light chain kinase steady‐state kinetics: comparison of smooth muscle myosin II and nonmuscle myosin IIB as substrates

Vascular smooth muscle myosin light chain diphosphorylation: Mechanism, function, and pathological implications

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