Substrate recognition by Ca2+/calmodulin-dependent protein kinase kinase -Role of the Arg-Pro-rich insert domain

Mammalian Ca2+/CaM-dependent protein kinase kinase (CaM-KK) has been identi fied and cloned as an activator for two kinases, CaM kinase I (CaM-KI) and CaM kinase IV (CaM-KIV), and a recent report (Yano, S., Togumitsu, Il., and Soderling, T. R. (1998) Nature 396, 584-587) demonstrates that CaM-KH can also activate and phosphorylate protein kinase B (PKB), In this study, we i dentify a CaM-KK from Caenorhabditis elegans, and comparison of its sequenc e with the mammalian CaM-KK alpha and beta shows a unique Arg-Pro (RP)-rich insert in their catalytic domains relative to other protein kinases, Delet ion of the RP-domain resulted in complete loss of CaM-KIV activation activi ty and physical interaction of CaM-KK with glutathione S-transferase-CaM-KI V (T196A). However, CaM-KK autophosphorylation and phosphorylation of a syn thetic peptide substrate were normal in the RP-domain mutant. Site-directed mutagenesis of three conserved Arg in the RP-domain of CaM-KK confirmed th at these positive charges are important for CaM-KIV activation. The RP-doma in deletion mutant also failed to fully activate and phosphorylate CaM-KI, but this mutant was indistinguishable from wild-type CaM-KK for the phospho rylation and activation of PKB, These results indicate that the RP-domain i n CaM-KK is critical for recognition of downstream CaM-kinases but not for its catalytic activity (i.e. autophosphorylation) and PKB activation.