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

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 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.

Molecular Mechanisms Underlying Ca2+/Calmodulin-Dependent Protein Kinase Kinase Signal Transduction

“…RP-domain deletion from CaMKKα’s catalytic domain impaired CaMKI and CaMKIV, but not PKB/Akt phosphorylation and activation, suggesting that the RP domain is involved in CaMKI and CaMKIV recognition as substrates [ 39 ]. A further study confirmed the requirement of the RP domain of both CaMKK isoforms for CaMKI, CaMKIV, and AMPK interaction and phosphorylation in vitro [ 65 ]. Interestingly, RP-sequence insertion between kinase subdomains II and III of the catalytic domain of liver kinase B1 (LKB1), an alternative activating kinase for AMPK incapable of phosphorylating CaMKI and CaMKIV, resulted in the acquisition of the CaMKIα- and CaMKIV-phosphorylating activity in the LKB1 mutants.…”

“…With increasing intracellular Ca 2+ concentration, Ca 2+ / CaM binds to regulatory domain of CaMKKα/1 ( E ) to suppress autoinhibition, thereby activating the kinase [ 64 ]. An activated CaMKK recognizes and phosphorylates downstream kinases including CaMKI, IV, and AMPK by using an Arg/Pro rich insert domain (RP-domain, D ) [ 39 , 65 ]. Amino acid sequence alignments of the regulatory domain including the autoinhibitory and Ca 2+ /CaM binding segments ( C ) and RP-domain ( D ) in various CaMKKs (rat, human α/1 and β/2 isoforms, and C. elegans ).…”

Development of a novel AAK1 inhibitor via Kinobeads-based screening

“…Expression plasmids for His 6 -tagged rat CaMKKα/1 (His-CaMKKα/1) and His 6 -tagged rat CaMKKβ/2 (His-CaMKKβ/2) were constructed using the pME-18s vector 33 . His-CaMKKα/1 and His-CaMKKβ/2 were expressed in COS-7 cells by transfection of both expression plasmids as described above, followed by purification with Ni-sepharose and CaM-sepharose resins.…”