The ubiquitin-associated domain of AMPK-related kinases regulates conformation and LKB1-mediated phosphorylation and activation

Abstract

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.

Conformational instability of the MARK3 UBA domain compromises ubiquitin recognition and promotes interaction with the adjacent kinase domain

“…These data indicate that stabilization of the atypical hMARK3 UBA domain fold by the kinase domain N-lobe interaction does not measurably increase the affinity of the hMARK3 UBA domain for Ub. Our findings are concordant with those of Jaleel et al (17), who used pull-down assays to demonstrate that the AMPK/Snf1 family UBA domains have no detectable affinity for different Ub linkages and Ub-like molecules in the context of isolated UBA domains and full-length kinases. These findings can be rationalized structurally by superimposing the canonical Dsk2p UBA domain:Ub complex structure on our hMARK3 kinase:UBA domain crystal structure (SI Fig.…”

“…It is noteworthy that with the exception of the MGY motif and hydrophobic core residues (SI Fig. 6) (16,17,20), UBA domains show little sequence conservation, and a tyrosine residue at the position homologous to Y361 of hMARK3 is seldom observed outside of the AMPK/Snf1 kinase family. Because of this poor sequence conservation, it therefore is difficult to predict whether a UBA domain is likely to adopt the topology identified in MARK kinases or a canonical fold.…”

“…With these factors in mind, we next examined whether the hMARK3 UBA domain possesses an intrinsic ability to bind Ub. Previous biochemical experiments suggest that the MARK UBA domains do not measurably interact with Ub and Ub-like molecules in vitro (17,20). Consequently, the MARK UBA domains can be categorized as one of the Ϸ30% of UBA domains that do not detectably bind Ub (16).…”

“…Previous studies have demonstrated the importance of the UBA domains within the Par1/MARKs and other AMPK/Snf1 family members for phosphorylation of the adjacent kinase domain's activation segment by the upstream activator complex, LKB1/ Par4:Mo25:STRAD (17). These data are consistent with a functional role for the UBA domains in stabilizing an open conformation of the N-and C-terminal lobes of the kinase domain through interactions with the N-lobe (as observed in the kinase:UBA domain crystal structures), which in turn exposes the kinase domain activation segment for phosphorylation by LKB1/Par4.…”

“…Predominantly, UBA domains have been characterized as binding monoUb and K48-linked and K63-linked polyUb (15,16), although a recent survey of 30 yeast and mammalian UBA domains revealed that Ϸ30% of UBA domains do not detectably interact with monoUb or polyUb chains in vitro (16). Further studies indicated that the MARK UBA domains belong to this latter class, as they were unable to detectably bind Ub or Ub-like species in vitro (17). However, comparison of the amino acid sequences of Ub-binding and nonbinding UBA domains did not show any obvious differences that would explain at a molecular level why a significant fraction of UBA domains fail to detectably bind Ub in vitro (16).…”