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

Riku Kaneshige, Satomi Ohtsuka, Yuhei Harada, Issei Kawamata, Masaki Magari, Naoki Kanayama, Naoya Hatano, Hiroyuki Sakagami, Hiroshi Tokumitsu

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)


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.

Original languageEnglish
JournalFEBS Journal
Publication statusAccepted/In press - 2022


  • AMP-activated protein kinase
  • Arg/Pro-rich insert domain (RP-domain)
  • calcium/calmodulin-dependent protein kinase
  • calcium/calmodulin-dependent protein kinase kinase
  • substrate recognition

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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