Jose M. Lizcano

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.

The insulin signalling pathway.

A key aim for future research is to identify the hydrophobic motif kinase that phosphorylates PKB, S6K, SGK and to establish how this protein kinase(s) is regulated by PtdIns(3,4,5)P3. It is also becoming obvious that the information obtained by the overexpression of constitutively active and dominant negative mutants of protein kinases is not providing physiologically reliable results and that new genetic and pharmacological approaches are needed to identify the substrates of each of the individual insulin-regulated protein kinases and to establish their roles in mediating insulin-dependent responses. Another key challenge will be to define the mechanism by which PKB and the activation of TC10 trigger the translocation of GLUT4 from its intracellular stores to the plasma membrane. Although our understanding of the insulin signal transduction pathway is far from being complete, our current knowledge of this pathway provides a framework for the development of novel drugs to treat diabetes. For example a drug that could mimic PtdIns(3,4,5)P3 would be expected to promote glucose uptake, glycogen synthesis and stimulate protein synthesis in tissues of diabetic patients. Recent studies have also demonstrated that inhibitors of GSK3, do indeed mimic the effects of insulin on this enzyme in cell lines and promote the uptake of glucose from the blood and its conversion to glycogen. If drugs that mimic the effect that insulin has on its signalling pathway are orally effective, it is possible that these could be used to treat type 1, as well as type 2 diabetes, and so reduce or replace the need for daily insulin injections.

High resolution crystal structure of the human PDK1 catalytic domain defines the regulatory phosphopeptide docking site

3‐phosphoinositide dependent protein kinase‐1 (PDK1) plays a key role in regulating signalling pathways by activating AGC kinases such as PKB/Akt and S6K. Here we describe the 2.0 Å crystal structure of the PDK1 kinase domain in complex with ATP. The structure defines the hydrophobic pocket termed the ‘PIF‐pocket’, which plays a key role in mediating the interaction and phosphorylation of certain substrates such as S6K1. Phosphorylation of S6K1 at its C‐terminal PIF‐pocket‐interacting motif promotes the binding of S6K1 with PDK1. In the PDK1 structure, this pocket is occupied by a crystallographic contact with another molecule of PDK1. Interestingly, close to the PIF‐pocket in PDK1, there is an ordered sulfate ion, interacting tightly with four surrounding side chains. The roles of these residues were investigated through a combination of site‐directed mutagenesis and kinetic studies, the results of which confirm that this region of PDK1 represents a phosphate‐dependent docking site. We discuss the possibility that an analogous phosphate‐binding regulatory motif may participate in the activation of other AGC kinases. Furthermore, the structure of PDK1 provides a scaffold for the design of specific PDK1 inhibitors.

Identification of the sucrose non-fermenting related kinase SNRK, as a novel LKB1 substrate

Recent work has shown that the LKB1 tumour suppressor protein kinase phosphorylates and activates protein kinases belonging to the AMP activated kinase (AMPK) subfamily. In this study, we identify the sucrose non‐fermenting protein (SNF1)‐related kinase (SNRK), a largely unstudied AMPK subfamily member, as a novel substrate for LKB1. We demonstrate that LKB1 activates SNRK by phosphorylating the T‐loop residue (Thr173), and that the LKB1 regulatory subunits STRAD and MO25 are required for LKB1 to activate SNRK. We find that SNRK is not active when expressed in HeLa cells that lack expression of LKB1, and its activity is restored by expression of wild type LKB1, but not catalytically deficient LKB1. We also present evidence that two other AMPK‐related kinases more distantly related to AMPK than SNRK, namely NIM1 and testis‐specific serine/threonine kinase‐1 (TSSK1) are not substrates for LKB1. Tissue distribution analysis indicates that SNRK protein is mainly expressed in testis, similar to TSSK isoforms, whereas NIM1 is more widely expressed. These results provide evidence that SNRK could mediate some of the physiological effects of LKB1.

Dopaminergic and Glutamatergic Signaling Crosstalk in Huntington's Disease Neurodegeneration: The Role of p25/Cyclin-Dependent Kinase 5

Altered glutamatergic and dopaminergic signaling has been proposed as contributing to the specific striatal cell death observed in Huntingtons disease (HD). However, the precise mechanisms by which mutant huntingtin sensitize striatal cells to dopamine and glutamate inputs remain unclear. Here, we demonstrate in knock-in HD striatal cells that mutant huntingtin enhances dopamine-mediated striatal cell death via dopamine D1 receptors. Moreover, we show that NMDA receptors specifically potentiate the vulnerability of mutant huntingtin striatal cells to dopamine toxicity as pretreatment with NMDA increased D1R-induced cell death in mutant but not wild-type cells. As potential underlying mechanism of increased striatal vulnerability, we identified aberrant cyclin-dependent kinase 5 (Cdk5) activation. We demonstrate that enhanced Cdk5 phosphorylation and increased calpain-mediated conversion of the Cdk5 activator p35 into p25 may account for the deregulation of Cdk5 associated to dopamine and glutamate receptor activation in knock-in HD striatal cells. Moreover, supporting a detrimental role of Cdk5 in striatal cell death, neuronal loss can be widely prevented by roscovitine, a potent Cdk5 inhibitor. Significantly, reduced Cdk5 expression together with enhanced Cdk5 phosphorylation and p25 accumulation also occurs in the striatum of mutant HdhQ111 mice and HD human brain suggesting the relevance of deregulated Cdk5 pathway in HD pathology. These findings provide new insights into the molecular mechanisms underlying the selective vulnerability of striatal cells in HD and identify p25/Cdk5 as an important mediator of dopamine and glutamate neurotoxicity associated to HD.

Molecular basis for the substrate specificity of NIMA-related kinase-6 (NEK6). Evidence that NEK6 does not phosphorylate the hydrophobic motif of ribosomal S6 protein kinase and serum- and glucocorticoid-induced protein kinase in vivo

The AGC family of protein kinases, which includes isoforms of protein kinase B (also known as Akt), ribosomal S6 protein kinase (S6K), and serum- and glucocorticoid-induced protein kinase (SGK) are activated in response to many extracellular signals and play key roles in regulating diverse cellular processes. They are activated by the phosphorylation of the T loop of their kinase domain by the 3-phosphoinositide-dependent protein kinase-1 and by phosphorylation of a residue located C-terminal to the kinase domain in a region termed the hydrophobic motif. Recent work has implicated the NIMA (never in mitosis, geneA)-related kinase-6 (NEK6) as the enzyme that phosphorylates the hydrophobic motif of S6K1 in vivo. Here we demonstrate that in addition to phosphorylating S6K1 and SGK1 at their hydrophobic motif, NEK6 also phosphorylates S6K1 at two other sites and phosphorylates SGK1 at one other site in vitro. Employing the Jerini pepSTAR method in combination with kinetic analysis of phosphorylation of variant peptides, we establish the key substrate specificity determinants for NEK6. Our analysis indicates that NEK6 has a strong preference for Leu 3 residues N-terminal to the site of phosphorylation. Its mutation to either Ile or Val severely reduced the efficacy with which NEK6-phosphorylated peptide substrates, and moreover, mutation of the equivalent Leu residue in S6K1 or SGK1 prevented phosphorylation of their hydrophobic motifs by NEK6 in vitro. However, these mutants of S6K1 or SGK1 still became phosphorylated at their hydrophobic motif following insulin-like growth factor-1 stimulation of transfected 293 cells. This study provides the first description of the basis for the substrate specificity of NEK6 and indicates that NEK6 is unlikely to be responsible for the IGF1-induced phosphorylation of the hydrophobic motif of S6K, SGK, and protein kinase B isoforms in vivo.

Inhibition of monoamine oxidase A and B activities by imidazol(ine)/guanidine drugs, nature of the interaction and distinction from I2-imidazoline receptors in rat liver

I2‐Imidazoline sites ([3H]‐idazoxan binding) have been identified on monoamine oxidase (MAO) and proposed to modulate the activity of the enzyme through an allosteric inhibitory mechanism ( Tesson et al., 1995 ). The main aim of this study was to assess the inhibitory effects and nature of the inhibition of imidazol(ine)/guanidine drugs on rat liver MAO‐A and MAO‐B isoforms and to compare their inhibitory potencies with their affinities for the sites labelled by [3H]‐clonidine in the same tissue. Competition for [3H]‐clonidine binding in rat liver mitochondrial fractions by imidazol(ine)/guanidine compounds revealed that the pharmacological profile of the interaction (2 ‐ styryl ‐ 2 ‐ imidazoline, LSL 61112>idazoxan>2 ‐ benzofuranyl ‐ 2 ‐ imidazoline, 2‐BFI=cirazoline>guanabenz>oxymetazoline>>clonidine) was typical of that for I2‐sites. Clonidine inhibited rat liver MAO‐A and MAO‐B activities with very low potency (IC50s: 700 μM and 6 mM, respectively) and displayed the typical pattern of competitive enzyme inhibition (Lineweaver‐Burk plots: increased Km and unchanged Vmax values). Other imidazol(ine)/guanidine drugs also were weak MAO inhibitors with the exception of guanabenz, 2‐BFI and cirazoline on MAO‐A (IC50s: 4–11 μM) and 2‐benzofuranyl‐2‐imidazol (LSL 60101) on MAO‐B (IC50: 16 μM). Idazoxan was a full inhibitor, although with rather low potency, on both MAO‐A and MAO‐B isoenzymes (IC50s: 280 μM and 624 μM, respectively). Kinetic analyses of MAO‐A inhibition by these drugs revealed that the interactions were competitive. For the same drugs acting on MAO‐B the interactions were of the mixed type inhibition (increased Km and decreased Vmax values), although the greater inhibitory effects on the apparent value of Vmax/Km than on the Vmax value indicated that the competitive element of the MAO‐B inhibition predominated. Competition for [3H]‐Ro 41‐1049 binding to MAO‐A or [3H]‐Ro 19‐6327 binding to MAO‐B in rat liver mitochondrial fractions by imidazol(ine)/guanidine compounds revealed that the drug inhibition constants (Ki values) were similar to the IC50 values displayed for the inhibition of MAO‐A or MAO‐B activities. In fact, very good correlations were obtained when the affinities of drugs at MAO‐A or MAO‐B catalytic sites were correlated with their potencies in inhibiting MAO‐A (r=0.92) or MAO‐B (r=0.99) activity. This further suggested a direct drug interaction with the catalytic sites of MAO‐A and MAO‐B isoforms. No significant correlations were found when the potencies of imidazol(ine)/guanidine drugs at the high affinity site (pKiH, nanomolar range) or the low‐affinity site (pKiL, micromolar range) of I2‐imidazoline receptors labelled with [3H]‐clonidine were correlated with the pIC50 values of the same drugs for inhibition of MAO‐A or MAO‐B activity. These discrepancies indicated that I2‐imidazoline receptors are not directly related to the site of action of these drugs on MAO activity in rat liver mitochondrial fractions. Although these studies cannot exclude the presence of additional binding sites on MAO that do not affect the activity of the enzyme, they would suggest that I2‐imidazoline receptors represent molecular species that are distinct from MAO.

Amine oxidase substrates mimic several of the insulin effects on adipocyte differentiation in 3T3 F442A cells.

We have previously reported that substrates of monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO) exert short-term insulin-like effects in rat adipocytes, such as stimulation of glucose transport. In the present work, we studied whether these substrates could also mimic long-term actions of insulin. Adipose differentiation of 3T3 F442A cells, which is highly insulin-dependent, served as a model to test the effects of sustained administration of amine oxidase substrates. Daily treatment of confluent cells with 0.75 mM tyramine (a substrate of MAO and SSAO) or benzylamine (a substrate of SSAO) over 1 week caused the acquisition of typical adipocyte morphology. The stimulation of protein synthesis and triacylglycerol accumulation caused by tyramine or benzylamine reached one half of that promoted by insulin. This effect was insensitive to pargyline (an MAO inhibitor), but was inhibited by semicarbazide (an SSAO inhibitor) and by N-acetylcysteine (an antioxidant agent), suggesting the involvement of the H(2)O(2) generated during SSAO-dependent amine oxidation. Chronic administration of amine oxidase substrates also induced the emergence of adipose conversion markers, such as aP2, glycerol-3-phosphate dehydrogenase, the glucose transporter GLUT4, and SSAO itself. Moreover, cells treated with amines acquired the same insulin sensitivity regarding glucose transport as adipocytes classically differentiated with insulin. In all, most of the adipogenic effects of amines were additive to insulin. Our data reveal that amine oxidase substrates partially mimic the adipogenic effect of insulin in cultured preadipocytes. Furthermore, they suggest that SSAO not only represents a novel late marker of adipogenesis, but could also be directly involved in the triggering of terminal adipocyte differentiation.

Neuregulin signaling on glucose transport in muscle cells

Neuregulin-1, a growth factor that potentiates myogenesis induces glucose transport through translocation of glucose transporters, in an additive manner to insulin, in muscle cells. In this study, we examined the signaling pathway required for a recombinant active neuregulin-1 isoform (rhHeregulin-β1, 177–244, HRG) to stimulate glucose uptake in L6E9 myotubes. The stimulatory effect of HRG required binding to ErbB3 in L6E9 myotubes. PI3K activity is required for HRG action in both muscle cells and tissue. In L6E9 myotubes, HRG stimulated PKBα, PKBγ, and PKCζ activities. TPCK, an inhibitor of PDK1, abolished both HRG- and insulin-induced glucose transport. To assess whether PKB was necessary for the effects of HRG on glucose uptake, cells were infected with adenoviruses encoding dominant negative mutants of PKBα. Dominant negative PKB reduced PKB activity and insulin-stimulated glucose transport but not HRG-induced glucose transport. In contrast, transduction of L6E9 myotubes with adenoviruses encoding a dominant negative kinase-inactive PKCζ abolished both HRG- and insulin-stimulated glucose uptake. In soleus muscle, HRG induced PKCζ, but not PKB phosphorylation. HRG also stimulated the activity of p70S6K, p38MAPK, and p42/p44MAPK and inhibition of p42/p44MAPK partially repressed HRG action on glucose uptake. HRG did not affect AMPKα1 or AMPKα2 activities. In all, HRG stimulated glucose transport in muscle cells by activation of a pathway that requires PI3K, PDK1, and PKCζ, but not PKB, and that shows cross-talk with the MAPK pathway. The PI3K, PDK1, and PKCζ pathway can be considered as an alternative mechanism, independent of insulin, to induce glucose uptake.

Insulin-induced Drosophila S6 kinase activation requires phosphoinositide 3-kinase and protein kinase B.

An important mechanism by which insulin regulates cell growth and protein synthesis is through activation of the p70 ribosomal S6 protein kinase (S6K). In mammalian cells, insulin-induced PI3K (phosphoinositide 3-kinase) activation, generates the lipid second messenger PtdIns(3,4,5) P (3), which is thought to play a key role in triggering the activation of S6K. Although the major components of the insulin-signalling pathway are conserved in Drosophila, recent studies suggested that S6K activation does not require PI3K in this system. To investigate further the role of dPI3K (Drosophila PI3K) in dS6K (Drosophila S6K) activation, we examined the effect of two structurally distinct PI3K inhibitors on insulin-induced dS6K activation in Kc167 and S2 Drosophila cell lines. We found that both inhibitors prevented insulin-stimulated phosphorylation and activation of dS6K. To investigate further the role of the dPI3K pathway in regulating dS6K activation, we also used dsRNAi (double-stranded RNA-mediated interference) to decrease expression of dPI3K and the PtdIns(3,4,5) P (3) phosphatase dPTEN ( Drosophila phosphatase and tensin homologue deleted on chromosome 10) in Kc167 and S2 cells. Knock-down of dPI3K prevented dS6K activation, whereas knock-down of dPTEN, which would be expected to increase PtdIns(3,4,5) P (3) levels, stimulated dS6K activity. Moreover, when the expression of the dPI3K target, dPKB (Drosophila protein kinase B), was decreased to undetectable levels, we found that insulin could no longer trigger dS6K activation. This observation provides the first direct demonstration that dPKB is required for insulin-stimulated dS6K activation. We also present evidence that the amino-acid-induced activation of dS6K in the absence of insulin, thought to be mediated by dTOR (Drosophila target of rapamycin), which is unaffected by the inhibition of dPI3K by wortmannin. The results of the present study support the view that, in Drosophila cells, dPI3K and dPKB, as well dTOR, are required for the activation of dS6K by insulin.