Calmodulin kinase II chimeras used to investigate the structural requirements for smooth muscle myosin light chain kinase autoinhibition and calmodulin-dependent activation.

Published

Journal Article

Segments of the autoregulatory domain of MK, a catalytically active fragment of the monomeric smooth muscle myosin light chain kinase (smMLCK) (residues 472-972), were replaced with their counterparts from a homologous but multimeric enzyme, calmodulin-dependent protein kinase II (CaM KII). Chimeric proteins in which both the autoregulatory and oligomerization domains of CaM KII (residues 281-478) were substituted for residues 781-972 of smMLCK, MK(CK281-478), or only the autoregulatory domain of CaM KII (residues 281-315) was exchanged for residues 781-813 of smMLCK, MK(CK281-315), exhibited significant enzymatic activity in the absence of Ca(2+)/CaM. In contrast, both MK and a chimeric protein in which the C-terminal half of the autoregulatory domain of smMLCK was replaced with CaM KII residues 301-315, MK(CK301-315), were inactive in the absence of Ca(2+)/CaM. These results indicate that the sequence of the N-terminal half of the autoregulatory domain of smMLCK is important for complete autoinhibition of its enzymatic activity. All proteins bound to Ca(2+)/CaM, and the chimeric proteins MK(CK281-478) and MK(CK281-315) were activated by Ca(2+)/CaM with activation constants (K(CaM)) and maximal enzymatic activities comparable to those of the wild-type MK enzyme. This demonstrates that the entire autoregulatory domain of CaM KII can replace that of smMLCK in its ability to promote efficient CaM-dependent activation of the smMLCK enzyme. However, the inability of the chimeric protein MK(CK301-315) to be activated by Ca(2+)/CaM suggests that replacement of only the C-terminal half of the autoregulatory domain of smMLCK, while still retaining the ability to bind Ca(2+)/CaM, also substitutes residues that prevent activation of the enzyme by Ca(2+)/CaM.

Full Text

Duke Authors

Cited Authors

  • Chin, D; Schreiber, JL; Means, AR

Published Date

  • November 1999

Published In

Volume / Issue

  • 38 / 46

Start / End Page

  • 15061 - 15069

PubMed ID

  • 10563788

Pubmed Central ID

  • 10563788

Electronic International Standard Serial Number (EISSN)

  • 1520-4995

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi990883a

Language

  • eng