Insulin activated endogenous protein kinase B alpha (also known as RAC/Akt kinase) activity 12‐fold in L6 myotubes, while after transfection into 293 cells PKBalpha was activated 20‐ and 50‐fold in response to insulin and IGF‐1 respectively. In both cells, the activation of PKBalpha was accompanied by its phosphorylation at Thr308 and Ser473 and, like activation, phosphorylation of both of these residues was prevented by the phosphatidylinositol 3‐kinase inhibitor wortmannin. Thr308 and/or Ser473 were mutated to Ala or Asp and activities of mutant PKBalpha molecules were analysed after transfection into 293 cells. The activity of wild‐type and mutant PKBalpha was also measured in vitro after stoichiometric phosphorylation of Ser473 by MAPKAP kinase‐2. These experiments demonstrated that activation of PKBalpha by insulin or insulin‐like growth factor‐1 (IGF‐1) results from phosphorylation of both Thr308 and Ser473, that phosphorylation of both residues is critical to generate a high level of PKBalpha activity and that the phosphorylation of Thr308 in vivo is not dependent on phosphorylation of Ser473 or vice versa. We propose a model whereby PKBalpha becomes phosphorylated and activated in insulin/IGF‐1‐stimulated cells by an upstream kinase(s).

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 450-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.

PDK1 is likely to mediate the activation of PKB by insulin or growth factors. DSTPK61 is a Drosophila homologue of PDK1. The effect of Ptdlns(3,4,5)P3/Ptdlns(3,4)P2 in the activation of PKB alpha is at least partly substrate directed.

Mutations in the human genes encoding WNK1 [with no K (lysine) protein kinase-1] and the related protein kinase WNK4 are the cause of Gordon's hypertension syndrome. Little is known about the molecular mechanism by which WNK isoforms regulate cellular processes. We immunoprecipitated WNK1 from extracts of rat testis and found that it was specifically associated with a protein kinase of the STE20 family termed 'STE20/SPS1-related proline/alanine-rich kinase' (SPAK). We demonstrated that WNK1 and WNK4 both interacted with SPAK as well as a closely related kinase, termed 'oxidative stress response kinase-1' (OSR1). Wildtype (wt) but not catalytically inactive WNK1 and WNK4 phosphorylated SPAK and OSR1 to a much greater extent than with other substrates utilized previously, such as myelin basic protein and claudin-4. Phosphorylation by WNK1 or WNK4 markedly increased SPAK and OSR1 activity. Phosphopeptide mapping studies demonstrated that WNK1 phosphorylated kinase-inactive SPAK and OSR1 at an equivalent residue located within the T-loop of the catalytic domain (Thr233 in SPAK, Thr185 in OSR1) and a serine residue located within a C-terminal non-catalytic region (Ser373 in SPAK, Ser325 in OSR1). Mutation of Thr185 to alanine prevented the activation of OSR1 by WNK1, whereas mutation of Thr185 to glutamic acid (to mimic phosphorylation) increased the basal activity of OSR1 over 20-fold and prevented further activation by WNK1. Mutation of Ser325 in OSR1 to alanine or glutamic acid did not affect the basal activity of OSR1 or its ability to be activated by WNK1. These findings suggest that WNK isoforms operate as protein kinases that activate SPAK and OSR1 by phosphorylating the T-loops of these enzymes, resulting in their activation. Our analysis also describes the first facile assay that can be employed to quantitatively assess WNK1 and WNK4 activity.

PDK1 is one of the components of the signaling pathway recruited by Pl 3-kinase for the activation of p70 S6 kinase as well as of PKB, and serves as a multifunctional effector downstream of the Pl 3-kinase.

Mutations increasing WNK1 kinase expression in humans cause the pseudohypoaldosteronism type II hypertension syndrome. This condition is treated effectively by thiazide diuretics, which exert their effects by inhibiting the Na+-Cl– cotransporter (NCC), suggesting a link between WNK1 and NCC. Here, we demonstrate that the SPAK and OSR1 kinases that are activated by WNK1 phosphorylate human NCC at three conserved residues (Thr46, Thr55 and Thr60). Activation of the WNK1-SPAK/OSR1 signalling pathway by treatment of HEK293 or mpkDCT kidney distal-convoluted-tubule-derived cells with hypotonic low-chloride conditions induced phosphorylation of NCC at residues phosphorylated by SPAK/OSR1. Efficient phosphorylation of NCC was dependent upon a docking interaction between an RFXI motif in NCC and SPAK/OSR1. Mutation of Thr60 to Ala in NCC markedly inhibited phosphorylation of Thr46 and Thr55 as well as NCC activation induced by hypotonic low-chloride treatment of HEK293 cells. Our results establish that the WNK1-SPAK/OSR1 signalling pathway plays a key role in controlling the phosphorylation and activity of NCC. They also suggest a mechanism by which increased WNK1 overexpression could lead to hypertension and that inhibitors of SPAK/OSR1 might be of use in reducing blood pressure by suppressing phosphorylation and hence activity of NCC.

Abbreviations used in this paper: FP, fl uorescent protein; SILAC, stable isotope labeling with amino acids in cell culture.The online version of this article contains supplemental material.

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 STRADa, a catalytically de®cient pseudokinase. Here we show that endogenous LKB1±STRADa complex is associated with a protein of unknown function, termed MO25a, through the interaction of MO25a with the last three residues of STRADa. MO25a and STRADa anchor LKB1 in the cytoplasm, excluding it from the nucleus. Moreover, MO25a enhances the formation of the LKB1±STRADa complex in vivo, stimulating the catalytic activity of LKB1~10-fold. We demonstrate that the related STRADb and MO25b 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.