Substrate recognition by Arg/Pro‐rich insert domain in calcium/calmodulin‐dependent protein kinase kinase for target protein kinases

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 Ca2+‐signaling pathways. Mammalian cells expressing CaMKKα and CaMKKβ lacking Arg/Pro‐rich insert domain (RP‐domain) sequences showed impaired phosphorylation of AMPKα, CaMKIα, 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 CaMKIα and CaMKIV; however, mutant LKB1 with the RP‐domain sequences of CaMKKα and CaMKKβ inserted between kinase subdomains II and III acquired CaMKIα and CaMKIV phosphorylating activity in vitro and in transfected cultured cells. Furthermore, ionomycin‐induced phosphorylation of hemagglutinin (HA)–CaMKIα at Thr177, HA–CaMKIV at Thr196, and HA–AMPKα 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 CaMKKα and CaMKKβ 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.