Calcium/calmodulin-dependent protein kinase kinases (CaMKKs) activate CaMKI, CaMKIV, protein kinase B/Akt, and AMP-activated protein kinase (AMPK) by phosphorylating Thr residues in activation loops to mediate various Ca 2+ -signaling pathways. Mammalian cells expressing CaMKKa and CaMKKb lacking Arg/Pro-rich insert domain (RP-domain) sequences showed impaired phosphorylation of AMPKa, CaMKIa, and CaMKIV, whereas the autophosphorylation activities of CaMKK mutants remained intact and were similar to those of wild-type CaMKKs. Liver kinase B1 (LKB1, an AMPK kinase) complexed with STRAD and MO25 and was unable to phosphorylate CaMKIa and CaMKIV; however, mutant LKB1 with the RP-domain sequences of CaMKKa and CaMKKb inserted between kinase subdomains II and III acquired CaMKIa and CaMKIV phosphorylating activity in vitro and in transfected cultured cells. Furthermore, ionomycin-induced phosphorylation of hemagglutinin (HA)-CaMKIa at Thr177, HA-CaMKIV at Thr196, and HA-AMPKa at Thr172 in transfected cells was significantly suppressed by cotransfection of kinase-dead mutants of CaMKK isoforms, but these dominant-negative effects were abrogated with RP-deletion mutants, suggesting that sequestration of substrate kinases by loss-of-function CaMKK mutants requires the RP-domain. This was confirmed by pulldown experiments that showed that dominant-negative mutants of CaMKKa and CaMKKb interact with target kinases but not RP-deletion mutants. Taken together, these results clearly indicate that both CaMKK isoforms require the RP-domain to recognize downstream kinases to interact with and phosphorylate Thr residues in their activation loops. Thus, the RP-domain may be a promising target for specific CaMKK inhibitors.