Regulatory mechanism of Ca²⁺/calmodulin-dependent protein kinase kinase

Ca²⁺/calmodulin-dependent protein kinase kinase (CaM-KK) is a novel member of the CaM kinase family, which specifically phosphorylates and activates CaM kinase I and IV. In this study, we characterized the CaM- binding peptide of αCaM-KK (residues 438-463), which suppressed the activity of constitutively active CaM-KK (84-434) in the absence of Ca²⁺/CaM but competitively with ATP. Truncation and Site-directed mutagenesis of the CaM- binding region in CaM-KK reveal that Ile⁴⁴¹ is essential for autoinhibition of CaM-KK. Furthermore, CaM-KK chimera mutants containing the CaM-binding sequence of either myosin light chain kinases or CaM kinase II located C-terminal of Leu⁴⁴⁰, exhibited enhanced Ca²⁺/CaM-independent activity (60% of total activity). Although the CaM-binding domains of myosin light chain kinases and CaM kinase II bind to the N- and C-terminal domains of CaM in the opposite orientation to CaM-KK (Osawa, M., Tokumitsu, H., Swindells, M. B., Kurihara, H., Orita, M., Shibanuma, T., Furuya, T., and Ikura, M. (1999) Nat. Struct. Biol. 6, 819-824), the chimeric CaM-KKs containing Ile⁴⁴¹ remained Ca²⁺/CaM-dependent. This result demonstrates that the orientation of the CaM binding is not critical for relief of CaM-KK autoinhibition. However, the requirement of Ile⁴⁴¹ for autoinhibition, which is located at the -3 position from the N-terminal anchoring residue (Trp⁴⁴⁴) to CaM, accounts for the opposite orientation of CaM binding of CaM-KK compared with other CaM kinases.