Photoaffinity labeling of a peptide substrate to myosin light chain kinase

Z. H. Gao, G. Zhi, B. P. Herring, C. Moomaw, L. Deogny, Clive A. Slaughter, J. T. Stull

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

The substrate binding properties of skeletal muscle myosin light chain kinase were investigated with a synthetic peptide containing the photoreactive amino acid p-benzoylphenylalanine (Bpa) incorporated amino- terminal of the phosphoacceptor serine (BpaKKRAARATSNVFA). When photolyzed at 350 nm, the peptide was cross-linked stoichiometrically to myosin light chain kinase in a Ca2+/calmodulin-dependent manner. Peptide incorporation into kinase inhibited light chain phosphorylation, and the loss of kinase activity was proportional to the extent of peptide incorporated. After peptide I was incorporated into myosin light chain kinase, it was partially phosphorylated in the absence of Ca2+/calmodulin. The extent of phosphorylation increased in the presence of Ca2+/calmodulin. The cross-linked photoadduct was digested, labeled peptides were purified by high performance liquid chromatography, and sites of covalent modification were determined by amino acid sequencing and analysis. The covalent modification in the catalytic core occurred on Ile-373 (66%) and in a peptide containing residues Asn-422 to Met-437 (14%), respectively. Lys-572 in the autoinhibitory region accounted for 20% of the incorporated label. The coincident covalent modification of the autoinhibitory domain suggests that it is located near the catalytic site. Based upon a model of the catalytic core, the substrate peptide is predicted to bind in the cleft between the two lobes of the kinase. The orientation of the substrate peptide on myosin light chain kinase is similar to the orientation of the substrate recognition fragment, but not the high affinity binding fragment, of inhibitor peptide of cAMP-dependent protein kinase in the catalytic subunit of the cAMP-dependent protein kinase.

Original languageEnglish (US)
Pages (from-to)10125-10135
Number of pages11
JournalJournal of Biological Chemistry
Volume270
Issue number17
DOIs
StatePublished - Jan 1 1995
Externally publishedYes

Fingerprint

Myosin-Light-Chain Kinase
Labeling
Peptides
Substrates
Catalytic Domain
Calmodulin
Phosphotransferases
Phosphorylation
Cyclic AMP-Dependent Protein Kinases
Skeletal Muscle Myosins
Peptide Fragments
Amino Acids
Protein Sequence Analysis
High performance liquid chromatography
Serine
Labels
High Pressure Liquid Chromatography
Light

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Gao, Z. H., Zhi, G., Herring, B. P., Moomaw, C., Deogny, L., Slaughter, C. A., & Stull, J. T. (1995). Photoaffinity labeling of a peptide substrate to myosin light chain kinase. Journal of Biological Chemistry, 270(17), 10125-10135. https://doi.org/10.1074/jbc.270.17.10125

Photoaffinity labeling of a peptide substrate to myosin light chain kinase. / Gao, Z. H.; Zhi, G.; Herring, B. P.; Moomaw, C.; Deogny, L.; Slaughter, Clive A.; Stull, J. T.

In: Journal of Biological Chemistry, Vol. 270, No. 17, 01.01.1995, p. 10125-10135.

Research output: Contribution to journalArticle

Gao, ZH, Zhi, G, Herring, BP, Moomaw, C, Deogny, L, Slaughter, CA & Stull, JT 1995, 'Photoaffinity labeling of a peptide substrate to myosin light chain kinase', Journal of Biological Chemistry, vol. 270, no. 17, pp. 10125-10135. https://doi.org/10.1074/jbc.270.17.10125
Gao, Z. H. ; Zhi, G. ; Herring, B. P. ; Moomaw, C. ; Deogny, L. ; Slaughter, Clive A. ; Stull, J. T. / Photoaffinity labeling of a peptide substrate to myosin light chain kinase. In: Journal of Biological Chemistry. 1995 ; Vol. 270, No. 17. pp. 10125-10135.
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