Jérôme Boudeau

LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1

We recently demonstrated that the LKB1 tumour suppressor kinase, in complex with the pseudokinase STRAD and the scaffolding protein MO25, phosphorylates and activates AMP‐activated protein kinase (AMPK). A total of 12 human kinases (NUAK1, NUAK2, BRSK1, BRSK2, QIK, QSK, SIK, MARK1, MARK2, MARK3, MARK4 and MELK) are related to AMPK. Here we demonstrate that LKB1 can phosphorylate the T‐loop of all the members of this subfamily, apart from MELK, increasing their activity >50‐fold. LKB1 catalytic activity and the presence of MO25 and STRAD are required for activation. Mutation of the T‐loop Thr phosphorylated by LKB1 to Ala prevented activation, while mutation to glutamate produced active forms of many of the AMPK‐related kinases. Activities of endogenous NUAK2, QIK, QSK, SIK, MARK1, MARK2/3 and MARK4 were markedly reduced in LKB1‐deficient cells. Neither LKB1 activity nor that of AMPK‐related kinases was stimulated by phenformin or AICAR, which activate AMPK. Our results show that LKB1 functions as a master upstream protein kinase, regulating AMPK‐related kinases as well as AMPK. Between them, these kinases may mediate the physiological effects of LKB1, including its tumour suppressor function.

MO25α/β interact with STRADα/β enhancing their ability to bind, activate and localize LKB1 in the cytoplasm

Mutations in the LKB1 protein kinase result in the inherited Peutz Jeghers cancer syndrome. LKB1 has been implicated in regulating cell proliferation and polarity although little is known about how this enzyme is regulated. We recently showed that LKB1 is activated through its interaction with STRADα, a catalytically deficient pseudokinase. Here we show that endogenous LKB1–STRADα complex is associated with a protein of unknown function, termed MO25α, through the interaction of MO25α with the last three residues of STRADα. MO25α and STRADα anchor LKB1 in the cytoplasm, excluding it from the nucleus. Moreover, MO25α enhances the formation of the LKB1–STRADα complex in vivo, stimulating the catalytic activity of LKB1 ∼10‐fold. We demonstrate that the related STRADβ and MO25β isoforms are also able to stabilize LKB1 in an active complex and that it is possible to isolate complexes of LKB1 bound to STRAD and MO25 isoforms, in which the subunits are present in equimolar amounts. Our results indicate that MO25 may function as a scaffolding component of the LKB1–STRAD complex and plays a crucial role in regulating LKB1 activity and cellular localization.

Identification of Protor as a novel Rictor-binding component of mTOR complex-2

The mTOR (mammalian target of rapamycin) protein kinase is an important regulator of cell growth. Two complexes of mTOR have been identified: complex 1, consisting of mTOR-Raptor (regulatory associated protein of mTOR)-mLST8 (termed mTORC1), and complex 2, comprising mTOR-Rictor (rapamycininsensitive companion of mTOR)-mLST8-Sin1 (termed mTORC2). mTORC1 phosphorylates the p70 ribosomal S6K (S6 kinase) at its hydrophobic motif (Thr389), whereas mTORC2 phosphorylates PKB (protein kinase B) at its hydrophobic motif (Ser473). In the present study, we report that widely expressed isoforms of unstudied proteins termed Protor-1 (protein observed with Rictor-1) and Protor-2 interact with Rictor and are components of mTORC2. We demonstrate that immunoprecipitation of Protor-1 or Protor-2 results in the co-immunoprecipitation of other mTORC2 subunits, but not Raptor, a specific component of mTORC1. We show that detergents such as Triton X-100 or n-octylglucoside dissociate mTOR and mLST8 from a complex of Protor-1, Sin1 and Rictor. We also provide evidence that Rictor regulates the expression of Protor-1, and that Protor-1 is not required for the assembly of other mTORC2 subunits into a complex. Protor-1 is a novel Rictor-binding subunit of mTORC2, but further work is required to establish its role.

Activation of the tumour suppressor kinase LKB1 by the STE20‐like pseudokinase STRAD

The LKB1 gene encodes a serine/threonine kinase mutated in Peutz–Jeghers cancer syndrome. Despite several proposed models for LKB1 function in development and in tumour suppression, the detailed molecular action of LKB1 remains undefined. Here, we report the identification and characterization of an LKB1‐specific adaptor protein and substrate, STRAD (STe20 Related ADaptor). STRAD consists of a STE20‐ like kinase domain, but lacks several residues that are indispensable for intrinsic catalytic activity. Endogenous LKB1 and STRAD form a complex in which STRAD activates LKB1, resulting in phosphorylation of both partners. STRAD determines the subcellular localization of wild‐type, but not mutant LKB1, translocating it from nucleus to cytoplasm. One LKB1 mutation previously identified in a Peutz–Jeghers family that does not compromise its kinase activity is shown here to interfere with LKB1 binding to STRAD, and hence with STRAD‐dependent regulation. Removal of endogenous STRAD by siRNA abrogates the LKB1‐induced G1 arrest. Our results imply that STRAD plays a key role in regulating the tumour suppressor activities of LKB1.

LKB1, a protein kinase regulating cell proliferation and polarity

LKB1 is a serine‐threonine protein kinase mutated in patients with an autosomal dominantly inherited cancer syndrome predisposing to multiple benign and malignant tumours, termed Peutz–Jeghers syndrome. Since its discovery in 1998, much research has focused on identification and characterisation of its cellular roles and analysing how LKB1 might be regulated. In this review we discuss exciting recent advances indicating that LKB1 functions as a tumour suppressor perhaps by controlling cell polarity. We also outline the current understanding of the molecular mechanisms by which LKB1 is regulated in vivo, through interaction with other proteins as well as by protein phosphorylation and prenylation.

Analysis of the LKB1-STRAD-MO25 complex

Mutations in the LKB1 tumour suppressor threonine kinase cause the inherited Peutz-Jeghers cancer syndrome and are also observed in some sporadic cancers. Recent work indicates that LKB1 exerts effects on metabolism, polarity and proliferation by phosphorylating and activating protein kinases belonging to the AMPK subfamily. In vivo, LKB1 forms a complex with STRAD, an inactive pseudokinase, and MO25, an armadillo repeat scaffolding-like protein. Binding of LKB1 to STRAD-MO25 activates LKB1 and re-localises it from the nucleus to the cytoplasm. To learn more about the inherent properties of the LKB1-STRAD-MO25 complex, we first investigated the activity of 34 point mutants of LKB1 found in human cancers and their ability to interact with STRAD and MO25. Interestingly, 12 of these mutants failed to interact with STRAD-MO25. Performing mutagenesis analysis, we defined two binding sites located on opposite surfaces of MO25α, which are required for the assembly of MO25α into a complex with STRADα and LKB1. In addition, we demonstrate that LKB1 does not require phosphorylation of its own T-loop to be activated by STRADα-MO25α, and discuss the possibility that this unusual mechanism of regulation arises from LKB1 functioning as an upstream kinase. Finally, we establish that STRADα, despite being catalytically inactive, is still capable of binding ATP with high affinity, but that this is not required for activation of LKB1. Taken together, our findings reinforce the functional importance of the binding of LKB1 to STRAD, and provide a greater understanding of the mechanism by which LKB1 is regulated and activated through its interaction with STRAD and MO25.

Regulation of activity and localization of the WNK1 protein kinase by hyperosmotic stress

Mutations within the WNK1 (with-no-K[Lys] kinase-1) gene cause Gordons hypertension syndrome. Little is known about how WNK1 is regulated. We demonstrate that WNK1 is rapidly activated and phosphorylated at multiple residues after exposure of cells to hyperosmotic conditions and that activation is mediated by the phosphorylation of its T-loop Ser382 residue, possibly triggered by a transautophosphorylation reaction. Activation of WNK1 coincides with the phosphorylation and activation of two WNK1 substrates, namely, the protein kinases STE20/SPS1-related proline alanine–rich kinase (SPAK) and oxidative stress response kinase-1 (OSR1). Small interfering RNA depletion of WNK1 impairs SPAK/OSR1 activity and phosphorylation of residues targeted by WNK1. Hyperosmotic stress induces rapid redistribution of WNK1 from the cytosol to vesicular structures that may comprise trans-Golgi network (TGN)/recycling endosomes, as they display rapid movement, colocalize with clathrin, adaptor protein complex 1 (AP-1), and TGN46, but not the AP-2 plasma membrane–coated pit marker nor the endosomal markers EEA1, Hrs, and LAMP1. Mutational analysis suggests that the WNK1 C-terminal noncatalytic domain mediates vesicle localization. Our observations shed light on the mechanism by which WNK1 is regulated by hyperosmotic stress.

ATP and MO25α Regulate the Conformational State of the STRADα Pseudokinase and Activation of the LKB1 Tumour Suppressor

The conformation of the pseudokinase STRADα, which is regulated by binding to ATP and to the scaffolding protein MO25α, is key to the activiation of the LKB1 tumor suppressor complex.

Heat-shock protein 90 and Cdc37 interact with LKB1 and regulate its stability.

LKB1 is a widely expressed serine/threonine protein kinase that is mutated in the inherited Peutz-Jeghers cancer syndrome. Recent findings indicate that LKB1 functions as a tumour suppressor, but little is known regarding the detailed mechanism by which LKB1 regulates cell growth. In this study we have purified LKB1 from cells and establish that it is associated with the heat-shock protein 90 (Hsp90) chaperone and the Cdc37 kinase-specific targetting subunit for Hsp90. We demonstrate that Cdc37 and Hsp90 bind specifically to the kinase domain of LKB1. We also perform experiments using Hsp90 inhibitors, which indicate that the association of Hsp90 and Cdc37 with LKB1 regulates LKB1 stability and prevents its degradation by the proteasome. Hsp90 inhibitors are being considered as potential anti-cancer agents. However, our observations indicate that prolonged usage of these drugs could possibly lead to tumour development by decreasing cellular levels of LKB1.

Identification and characterization of four novel phosphorylation sites (Ser31, Ser325, Thr336 and Thr366) on LKB1/STK11, the protein kinase mutated in Peutz-Jeghers cancer syndrome.

Peutz-Jeghers syndrome is an inherited cancer syndrome, which results in a greatly increased risk of developing tumours in those affected. The causative gene encodes a nuclear-localized protein kinase, termed LKB1, which is predicted to function as a tumour suppressor. The mechanism by which LKB1 is regulated in cells is not known, and nor have any of its physiological substrates been identified. Recent studies have demonstrated that LKB1 is phosphorylated in cells. As a first step towards identifying the roles that phosphorylation of LKB1 play, we have mapped the residues that are phosphorylated in human embryonic kidney (HEK)-293 cells, as well as the major in vitro autophosphorylation sites. We demonstrate that LKB1 expressed in HEK-293 cells, in addition to being phosphorylated at Ser(431), a previously characterized phosphorylation site, is also phosphorylated at Ser(31), Ser(325) and Thr(366). Incubation of wild-type LKB1, but not a catalytically inactive mutant, with manganese-ATP in vitro resulted in the phosphorylation of LKB1 at Thr(336) as well as at Thr(366). We were unable to detect autophosphorylation at Thr(189), a site previously claimed to be an LKB1 autophosphorylation site. A catalytically inactive mutant of LKB1 was phosphorylated at Ser(31) and Ser(325) in HEK-293 cells to the same extent as the wild-type enzyme, indicating that LKB1 does not phosphorylate itself at these residues. We show that phosphorylation of LKB1 does not directly affect its nuclear localization or its catalytic activity in vitro, but that its phosphorylation at Thr(336), and perhaps to a lesser extent at Thr(366), inhibits LKB1 from suppressing cell growth.