Calcium/calmodulin-dependent protein kinase kinase: Identification of regulatory domains

Hiroshi Tokumitsu, Gary A. Wayman, Masaaki Muramatsu, Thomas R. Soderling

    Research output: Contribution to journalArticle

    43 Scopus citations

    Abstract

    We recently cloned a calmodulin-dependent protein kinase kinase (CaM- KK) which phosphorylates and activates CaM-KI and CaM-KIV [Tokumitsu, H., Enslen, H., and Soderling, T. R. (1995) J. Biol. Chem. 270, 19320-19324]. In the present study, we have identified its regulatory CaM-binding and autoinhibitory domains (CBD and AID, respectively) using a series of COOH- terminal truncations and site-directed mutants expressed in COS-7 cells. Truncation mutant CaM-KK1-463 activated CaM-KIV and bound CaM similar to wild-type enzyme (CaM-KK1-505); CaM-KK1-448 did not bind CaM and was largely inactive; and CaM-KK1-434 also did not bind CaM but activated a CaM-independent mutant of CaM-KIV in the absence of Ca2+/CaM. Substitution of triple negative charges (Asp) at positions 455RKR, 448ILV, or 443SWT blocked CaM binding and suppressed by 70-90% CaM-KK activities. Mutants 438VKL and 435KNS to DDD exhibited partial Ca2+/CaM-independent activities. These results identify overlapping AID and CBD between residues 430 and 460 in CaM-KK, similar to other CaM-Ks. Consistent with this assignment, the synthetic peptide corresponding to residues 438-463 bound CaM in a Ca2+dependent manner with a K(d) in the low nanomolar range. Furthermore, phosphorylation by cAMP-kinase of Ser458 at the COOH-terminus of the CBD in CaM-KK, which suppresses subsequent CaM binding [Wayman, G., Tokumitsu, H., and Soderling, T. R. (1997) J. Biol. Chem. 272, 16073-16076], was blocked by prior binding of Ca2+/CaM to CaM- KK.

    Original languageEnglish (US)
    Pages (from-to)12823-12827
    Number of pages5
    JournalBiochemistry
    Volume36
    Issue number42
    DOIs
    StatePublished - Oct 21 1997

      Fingerprint

    ASJC Scopus subject areas

    • Biochemistry

    Cite this

    Tokumitsu, H., Wayman, G. A., Muramatsu, M., & Soderling, T. R. (1997). Calcium/calmodulin-dependent protein kinase kinase: Identification of regulatory domains. Biochemistry, 36(42), 12823-12827. https://doi.org/10.1021/bi971348i