Nick A. Morrice

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.

Inhibition of caspase-9 through phosphorylation at Thr 125 by ERK MAPK

Many pro-apoptotic signals activate caspase-9, an initiator protease that activates caspase-3 and downstream caspases to initiate cellular destruction. However, survival signals can impinge on this pathway and suppress apoptosis. Activation of the Ras–Raf–MEK–ERK mitogen-activated protein kinase (MAPK) pathway is associated with protection of cells from apoptosis and inhibition of caspase-3 activation, although the targets are unknown. Here, we show that the ERK MAPK pathway inhibits caspase-9 activity by direct phosphorylation. In mammalian cell extracts, cytochrome c-induced activation of caspases-9 and -3 requires okadaic-acid-sensitive protein phosphatase activity. The opposing protein kinase activity is overcome by treatment with the broad-specificity kinase inhibitor staurosporine or with inhibitors of MEK1/2. Caspase-9 is phosphorylated at Thr 125, a conserved MAPK consensus site targeted by ERK2 in vitro, in a MEK-dependent manner in cells stimulated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Phosphorylation at Thr 125 is sufficient to block caspase-9 processing and subsequent caspase-3 activation. We suggest that phosphorylation and inhibition of caspase-9 by ERK promotes cell survival during development and tissue homeostasis. This mechanism may also contribute to tumorigenesis when the ERK MAPK pathway is constitutively activated.

Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments.

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.

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.

Activation of the thiazide-sensitive Na + -Cl - cotransporter by the WNK-regulated kinases SPAK and OSR1

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.

GSK-3 phosphorylation of the Alzheimer epitope within collapsin response mediator proteins regulates axon elongation in primary neurons.

Elevated glycogen synthase kinase-3 (GSK-3) activity is associated with Alzheimer disease. We have found that collapsin response mediator proteins (CRMP) 2 and 4 are physiological substrates of GSK-3. The amino acids targeted by GSK-3 comprise a hyperphosphorylated epitope first identified in plaques isolated from Alzheimer brain. Expression of wild type CRMP2 in primary hippocampal neurons or SH-SY5Y neuroblastoma cells promotes axon elongation. However, a GSK-3-insensitive CRMP2 mutant has dramatically reduced ability to promote axon elongation, a similar effect to pharmacological inhibition of GSK-3. Hence, we propose that phosphorylation of CRMP proteins by GSK-3 regulates axon elongation. This work provides a direct connection between hyperphosphorylation of these residues and elevated GSK-3 activity, both of which are observed in Alzheimer brain.

Functional interactions of the SPAK/OSR1 kinases with their upstream activator WNK1 and downstream substrate NKCC1

The SPAK (STE20/SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase-1) kinases interact and phosphorylate NKCC1 (Na+-K+-2Cl- co-transporter-1), leading to its activation. Recent studies indicated that SPAK and OSR1 are phosphorylated and activated by the WNK1 [with no K (lysine) protein kinase-1] and WNK4, genes mutated in humans affected by Gordons hypertension syndrome. In the present study, we have identified three residues in NKCC1 (Thr175/Thr179/Thr184 in shark or Thr203/Thr207/Thr212 in human) that are phosphorylated by SPAK and OSR1, and have developed a peptide substrate, CATCHtide (cation chloride co-transporter peptide substrate), to assess SPAK and OSR1 activity. Exposure of HEK-293 (human embryonic kidney) cells to osmotic stress, which leads to phosphorylation and activation of NKCC1, increased phosphorylation of NKCC1 at the sites targeted by SPAK/OSR1. The residues on NKCC1, phosphorylated by SPAK/OSR1, are conserved in other cation co-transporters, such as the Na+-Cl- co-transporter, the target of thiazide drugs that lower blood pressure in humans with Gordons syndrome. Furthermore, we characterize the properties of a 92-residue CCT (conserved C-terminal) domain on SPAK and OSR1 that interacts with an RFXV (Arg-Phe-Xaa-Val) motif present in the substrate NKCC1 and its activators WNK1/WNK4. A peptide containing the RFXV motif interacts with nanomolar affinity with the CCT domains of SPAK/OSR1 and can be utilized to affinity-purify SPAK and OSR1 from cell extracts. Mutation of the arginine, phenylalanine or valine residue within this peptide abolishes binding to SPAK/OSR1. We have identified specific residues within the CCT domain that are required for interaction with the RFXV motif and have demonstrated that mutation of these in OSR1 inhibited phosphorylation of NKCC1, but not of CATCHtide which does not possess an RFXV motif. We establish that an intact CCT domain is required for WNK1 to efficiently phosphorylate and activate OSR1. These data establish that the CCT domain functions as a multipurpose docking site, enabling SPAK/OSR1 to interact with substrates (NKCC1) and activators (WNK1/WNK4).

14-3-3s regulate global cleavage of their diverse binding partners in sugar-starved Arabidopsis cells.

Despite 14‐3‐3 proteins being implicated in the control of the eukaryotic cell cycle, metabolism, cell signalling and survival, little is known about the global regulation or functions of the phosphorylation‐dependent binding of 14‐3‐3s to diverse target proteins. We identified Arabidopsis cytosolic proteins that bound 14‐3‐3s in competition with a 14‐3‐3‐binding phosphopeptide, including nitrate reductase, glyceraldehyde‐ 3‐phosphate dehydrogenase, a calcium‐dependent protein kinase, sucrose‐phosphate synthase (SPS) and glutamyl‐tRNA synthetase. Remarkably, in cells starved of sugars or fed with non‐metabolizable glucose analogues, all 14‐3‐3 binding was lost and the target proteins were selectively cleaved into proteolytic fragments. 14‐3‐3 binding reappeared after several hours of re‐feeding with sugars. Starvation‐induced degradation was blocked by 5‐amino imidazole‐4‐carboxamide riboside (which is converted to an AMP‐mimetic) or the protease inhibitor MG132 (Cbz‐leu‐leu‐leucinal). Extracts of sugar‐starved (but not sugar‐fed) Arabidopsis cells contained an ATP‐independent, MG132‐sensitive, neutral protease that cleaved Arabidopsis SPS, and the mammalian 14‐3‐3‐regulated transcription factor, FKHR. Cleavage of SPS and phosphorylated FKHR in vitro was blocked by binding to 14‐3‐3s. The finding that 14‐3‐3s participate in a nutrient‐sensing pathway controlling cleavage of many targets may underlie the effects of these proteins on plant development.

The Mitogen-Activated Protein Kinase Signal-Integrating Kinase Mnk2 Is a Eukaryotic Initiation Factor 4E Kinase with High Levels of Basal Activity in Mammalian Cells

ABSTRACT The cap-binding translation initiation factor eukaryotic initiation factor 4E (eIF4E) is phosphorylated in vivo at Ser209 in response to a variety of stimuli. In this paper, we show that the mitogen-activated protein kinase (MAPK) signal-integrating kinase Mnk2 phosphorylates eIF4E at this residue. Mnk2 binds to the scaffolding protein eIF4G, and overexpression of Mnk2 results in increased phosphorylation of endogenous eIF4E, showing that it can act as an eIF4E kinase in vivo. We have identified eight phosphorylation sites in Mnk2, of which at least three potential MAPK sites are likely to be essential for Mnk2 activity. In contrast to that of Mnk1, the activity of overexpressed Mnk2 is high under control conditions and could only be reduced substantially by a combination of PD98059 and SB203580, while the activity of endogenous Mnk2 in Swiss 3T3 cells was hardly affected upon treatment with these inhibitors. These compounds did not abolish phosphorylation of eIF4E, implying that Mnk2 may mediate phosphorylation of eIF4E in Swiss 3T3 cells. In vitro phosphorylation studies show that Mnk2 is a significantly better substrate than Mnk1 for extracellular signal-regulated kinase 2 (ERK2), p38MAPKα, and p38MAPKβ. Therefore, the high levels of activity of Mnk2 under several conditions may be explained by efficient activation of Mnk2 by low levels of activity of the upstream kinases. Interestingly, we found that the association of both Mnk1 and Mnk2 with eIF4G increased upon inhibition of the MAPK pathways while activation of ERK resulted in decreased binding to eIF4G. This might reflect a mechanism to ensure rapid, but transient, phosphorylation of eIF4E upon stimulation of the MAPK pathways.