Regulation of calcineurin by phosphorylation. Identification of the regulatory site phosphorylated by Ca2+/calmodulin-dependent protein kinase II and protein kinase C

Y. Hashimoto, Thomas Soderling

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The site in calcineurin, the Ca2+/calmodulin (CaM)-dependent protein phosphatase, which is phosphorylated by Ca2+/CaM-dependent protein kinase II (CaM-kinase II) has been identified. Analyses of 32P release from tryptic and cyanogen bromide peptides derived from [32P]calcineurin plus direct sequence determination established the site as -Arg-Val-Phe-Ser(PO4)-Val-Leu-Arg-, which conformed to the consensus phosphorylation sequence for CaM-kinase II (Arg-X-X-Ser/Thr-). This phosphorylation site is located at the C-terminal boundary of the putative CaM-binding domain in calcineurin (Kincaid, R.L., Nightingale, M.S., and Martin, B.M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8983-8987), thereby accounting for the observed inhibition of this phosphorylation when Ca2+/CaM is bound to calcineurin. Since the phosphorylation site sequence also contains elements of the specificity determinants for Ca2+/phospholipid-dependent protein kinase (protein kinase C) (basic residues both N-terminal and C-terminal to Ser/Thr), we tested calcineurin as a substrate for protein kinase C. Protein kinase C catalyzed rapid stoichiometric phosphorylation, and the characteristics of the reaction were the same as with CaM-kinase II: 1) the phosphorylation was blocked by binding of Ca2+/CaM to calcineurin; 2) phosphorylation partially inactivated calcineurin by increasing the K(m) (from 9.9 ± 1.1 to 17.5 ± 1.1 μM 32P-labeled myosin light chain); and 3) [32P]calcineurin exhibited very slow autodephosphorylation but was rapidly dephosphorylated by protein phosphatase IIA. Tryptic and thermolytic 32P-peptide mapping and sequential phosphoamino acid sequence analysis confirmed that protein kinase C and CaM-kinase II phosphorylated the same site.

Original languageEnglish (US)
Pages (from-to)16524-16529
Number of pages6
JournalJournal of Biological Chemistry
Issue number28
Publication statusPublished - 1989
Externally publishedYes


ASJC Scopus subject areas

  • Biochemistry

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