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Substrate recognition by Arg/Pro-rich insert domain in calcium/calmodulin-dependent protein kinase kinase for target protein kinases.
- Source :
-
The FEBS journal [FEBS J] 2022 Oct; Vol. 289 (19), pp. 5971-5984. Date of Electronic Publication: 2022 May 17. - Publication Year :
- 2022
-
Abstract
- 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 <superscript>2+</superscript> -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.<br /> (© 2022 Federation of European Biochemical Societies.)
- Subjects :
- AMP-Activated Protein Kinases genetics
AMP-Activated Protein Kinases metabolism
Animals
Calcium metabolism
Hemagglutinins
Ionomycin
Mammals metabolism
Phosphorylation
Protein Isoforms metabolism
Calcium-Calmodulin-Dependent Protein Kinase Kinase chemistry
Calcium-Calmodulin-Dependent Protein Kinase Kinase genetics
Proto-Oncogene Proteins c-akt metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1742-4658
- Volume :
- 289
- Issue :
- 19
- Database :
- MEDLINE
- Journal :
- The FEBS journal
- Publication Type :
- Academic Journal
- Accession number :
- 35490408
- Full Text :
- https://doi.org/10.1111/febs.16467