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Intrasteric regulation of myosin light chain kinase: the pseudosubstrate prototope binds to the active site.

Publication ,  Journal Article
Bagchi, IC; Kemp, BE; Means, AR
Published in: Mol Endocrinol
April 1992

We previously proposed a molecular mechanism for the activation of smooth muscle myosin light chain kinase (smMLCK) by calmodulin (CaM). According to this model, smMLCK is autoinhibited in the absence of Ca2+/CaM due to the interaction of a pseudosubstrate prototope, contained within the CaM binding/regulatory region, with the active site of the enzyme. Binding of Ca2+/CaM releases the autoinhibition and allows access of the protein substrate to the active site of the enzyme, resulting in phosphorylation of the myosin light chains. We now provide direct experimental evidence that the pseudosubstrate prototope can associate with the active site. We constructed a smMLCK mutant in which the five-amino acid phosphorylation site of the myosin light chain substrate was inserted into the pseudosubstrate sequence of the CaM binding domain without disrupting the ability of the enzyme to bind Ca2+/CaM. We demonstrate that this mutant undergoes intramolecular autophosphorylation at the appropriate inserted serine residue in the absence of CaM and that this autophosphorylation activates the enzyme. Binding of Ca2+/CaM to the mutant enzyme stimulated myosin light chain substrate phosphorylation but strongly inhibited autophosphorylation, presumably by removing the pseudosubstrate from the active site. These results confirm that the pseudosubstrate sequence has access to the catalytic site and that the activation of the enzyme is accompanied by its removal from this position due to Ca2+/CaM binding as predicted by the model.

Duke Scholars

Published In

Mol Endocrinol

DOI

ISSN

0888-8809

Publication Date

April 1992

Volume

6

Issue

4

Start / End Page

621 / 626

Location

United States

Related Subject Headings

  • Recombinant Proteins
  • Phosphorylation
  • Myosin-Light-Chain Kinase
  • Mutagenesis, Site-Directed
  • Muscle, Smooth
  • Molecular Weight
  • Molecular Sequence Data
  • Kinetics
  • Homeostasis
  • Gizzard, Avian
 

Citation

APA
Chicago
ICMJE
MLA
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Bagchi, I. C., Kemp, B. E., & Means, A. R. (1992). Intrasteric regulation of myosin light chain kinase: the pseudosubstrate prototope binds to the active site. Mol Endocrinol, 6(4), 621–626. https://doi.org/10.1210/mend.6.4.1584224
Bagchi, I. C., B. E. Kemp, and A. R. Means. “Intrasteric regulation of myosin light chain kinase: the pseudosubstrate prototope binds to the active site.Mol Endocrinol 6, no. 4 (April 1992): 621–26. https://doi.org/10.1210/mend.6.4.1584224.
Bagchi, I. C., et al. “Intrasteric regulation of myosin light chain kinase: the pseudosubstrate prototope binds to the active site.Mol Endocrinol, vol. 6, no. 4, Apr. 1992, pp. 621–26. Pubmed, doi:10.1210/mend.6.4.1584224.

Published In

Mol Endocrinol

DOI

ISSN

0888-8809

Publication Date

April 1992

Volume

6

Issue

4

Start / End Page

621 / 626

Location

United States

Related Subject Headings

  • Recombinant Proteins
  • Phosphorylation
  • Myosin-Light-Chain Kinase
  • Mutagenesis, Site-Directed
  • Muscle, Smooth
  • Molecular Weight
  • Molecular Sequence Data
  • Kinetics
  • Homeostasis
  • Gizzard, Avian