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STE20 phosphorylation of AMPK-related kinases revealed by biochemical purifications combined with genetics.

ABSTRACT: We have recently purified mammalian sterile 20 (STE20)-like kinase 3 (MST3) as a kinase for the multifunctional kinases, AMP-activated protein kinase-related kinases (ARKs). However, unresolved questions from this study, such as remaining phosphorylation activities following deletion of the Mst3 gene from human embryonic kidney cells and mice, led us to conclude that there were additional kinases for ARKs. Further purification recovered Ca²⁺/calmodulin-dependent protein kinase kinases 1 and 2 (CaMKK1 and 2), and a third round of purification revealed mitogen-activated protein kinase kinase kinase kinase 5 (MAP4K5) as potential kinases of ARKs. We then demonstrated that MST3 and MAP4K5, both belonging to the STE20-like kinase family, could phosphorylate all 14 ARKs both in vivo and in vitro. Further examination of all 28 STE20 kinases detected variable phosphorylation activity on AMP-activated protein kinase (AMPK) and the salt-inducible kinase 3 (SIK3). Taken together, our results have revealed novel relationships between STE20 kinases and ARKs, with potential physiological and pathological implications.

SUBMITTER: Liu Y

PROVIDER: S-EPMC9112000 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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STE20 phosphorylation of AMPK-related kinases revealed by biochemical purifications combined with genetics.

Liu Yuxiang Y Wang Tao V TV Cui Yunfeng Y Li Chaoyi C Jiang Lifen L Rao Yi Y

The Journal of biological chemistry 20220410 5

We have recently purified mammalian sterile 20 (STE20)-like kinase 3 (MST3) as a kinase for the multifunctional kinases, AMP-activated protein kinase-related kinases (ARKs). However, unresolved questions from this study, such as remaining phosphorylation activities following deletion of the Mst3 gene from human embryonic kidney cells and mice, led us to conclude that there were additional kinases for ARKs. Further purification recovered Ca<sup>2+</sup>/calmodulin-dependent protein kinase kinases ...[more]

PMID: 35413284

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Project description:Recent work indicates that the LKB1 tumour suppressor protein kinase, which is mutated in Peutz-Jeghers cancer syndrome, phosphorylates and activates a group of protein kinases that are related to AMPK (AMP-activated protein kinase). Ten of the 14 AMPK-related protein kinases activated by LKB1, including SIK (salt-induced kinase), MARK (microtubule-affinity-regulating kinase) and BRSK (brain-specific kinase) isoforms, possess a ubiquitin-associated (UBA) domain immediately C-terminal to the kinase catalytic domain. These are the only protein kinases in the human genome known to possess a UBA domain, but their roles in regulating AMPK-related kinases are unknown. We have investigated the roles that the UBA domain may play in regulating these enzymes. Limited proteolysis of MARK2 revealed that the kinase and UBA domains were contained within a fragment that was resistant to trypsin proteolysis. SAXS (small-angle X-ray scattering) analysis of inactive and active LKB1-phosphorylated MARK2 revealed that activation of MARK2 is accompanied by a significant conformational change that alters the orientation of the UBA domain with respect to the catalytic domain. Our results indicate that none of the UBA domains found in AMPK-related kinases interact with polyubiquitin or other ubiquitin-like molecules. Instead, the UBA domains appear to play an essential conformational role and are required for the LKB1-mediated phosphorylation and activation of AMPK-related kinases. This is based on the findings that mutation or removal of the UBA domains of several AMPK-related kinases, including isoforms of MARK, SIK and BRSK, markedly impaired the catalytic activity and LKB1-mediated phosphorylation of these enzymes. We also provide evidence that the UBA domains do not function as LKB1-STRAD (STE20-related adaptor)-MO25 (mouse protein 25) docking/interacting sites and that mutations in the UBA domain of SIK suppressed the ability of SIK to localize within punctate regions of the nucleus. Taken together, these findings suggest that the UBA domains of AMPK-related kinases play an important role in regulating the conformation, activation and localization of these enzymes.

Dataset Information

STE20 phosphorylation of AMPK-related kinases revealed by biochemical purifications combined with genetics.

Publications

STE20 phosphorylation of AMPK-related kinases revealed by biochemical purifications combined with genetics.

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