Studies of the regulatory mechanism of Ca2+/calmodulin-dependent protein kinase II. Mutation of threonine 286 to alanine and aspartate

Y. L. Fong, W. L. Taylor, A. R. Means, Thomas Soderling

Research output: Contribution to journalArticle

130 Citations (Scopus)

Abstract

A cDNA clone for the α subunit of mouse brain Ca2+/CaM-dependent protein kinase II (CaM-kinase II) was transcribed in vitro and translated in a rabbit reticulocyte lysate system. Inclusion of [35S]methionine in the translation system yielded a single 35S-polypeptide of about 50 kDa. When the translation system was assayed for CaM-kinase II activity, there was a 5-10-fold enrichment of kinase activity which was totally dependent on Ca2+/calmodulin (CaM). Both the 50-kDa 35S-polypeptide and the Ca2+/CaM-dependent protein kinase activity were quantitatively immunoprecipitated by rat brain CaM-kinase II antibody. When the translated wild-type kinase was subjected to autophosphorylation conditions in the presence of Ca2+, CaM, Mg2+, and ATP, the Ca2+-independent activity (assayed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid) increased from 5.8 ± 0.7 to 26.5 ± 2.1% of total activity (assayed in the presence of Ca2+/CaM). These properties confirm the identity of the kinase translated in vitro as CaM-kinase II. The role of Thr-286 autophosphorylation in formation of the Ca2+-independent activity was investigated by site-directed mutation of Thr-286 to Ala (Ala-286 kinase) and to Asp (Asp-286 kinase). The Ala-286 kinase was completely dependent on Ca2+/CaM for activity prior and subsequent to autophosphorylation. The Asp-286 kinase exhibited 21.9 ± 0.8% Ca2+-independent activity, and this was not increased by autophosphorylation. These results establish that introduction of negative charge(s) at residue 286, either by autophosphorylation of Thr or by mutation to Asp, is sufficient and necessary to generate the partially Ca2+-independent form of CaM-kinase II.

Original languageEnglish (US)
Pages (from-to)16759-16763
Number of pages5
JournalJournal of Biological Chemistry
Volume264
Issue number28
StatePublished - 1989
Externally publishedYes

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Calcium-Calmodulin-Dependent Protein Kinase Type 2
Threonine
Aspartic Acid
Alanine
Phosphotransferases
Mutation
Calmodulin
Protein Kinases
Brain
Calcium-Calmodulin-Dependent Protein Kinases
Peptides
Reticulocytes
Viperidae
Methionine
Rats
Complementary DNA
Clone Cells
Adenosine Triphosphate
Rabbits
Acids

ASJC Scopus subject areas

  • Biochemistry

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Studies of the regulatory mechanism of Ca2+/calmodulin-dependent protein kinase II. Mutation of threonine 286 to alanine and aspartate. / Fong, Y. L.; Taylor, W. L.; Means, A. R.; Soderling, Thomas.

In: Journal of Biological Chemistry, Vol. 264, No. 28, 1989, p. 16759-16763.

Research output: Contribution to journalArticle

Fong, Y. L. ; Taylor, W. L. ; Means, A. R. ; Soderling, Thomas. / Studies of the regulatory mechanism of Ca2+/calmodulin-dependent protein kinase II. Mutation of threonine 286 to alanine and aspartate. In: Journal of Biological Chemistry. 1989 ; Vol. 264, No. 28. pp. 16759-16763.
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