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

Autor: Kaneshige R; Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, Japan., Ohtsuka S; Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, Japan., Harada Y; Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, Japan., Kawamata I; Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Okayama University, Japan., Magari M; Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, Japan., Kanayama N; Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, Japan., Hatano N; Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, Japan., Sakagami H; Department of Anatomy, Kitasato University School of Medicine, Sagamihara, Japan., Tokumitsu H; Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, Japan.
Jazyk: angličtina
Zdroj: The FEBS journal [FEBS J] 2022 Oct; Vol. 289 (19), pp. 5971-5984. Date of Electronic Publication: 2022 May 17.
DOI: 10.1111/febs.16467
Abstrakt: 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 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.
(© 2022 Federation of European Biochemical Societies.)
Databáze: MEDLINE
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