InteractiveFly: GeneBrief

Lk6 kinase: Biological Overview | References

BIOLOGICAL OVERVIEW

Eukaryotic initiation factor 4E (eIF4E) controls a crucial step of translation initiation and is critical for cell growth. Biochemical studies have shown that it undergoes a regulated phosphorylation by the MAP-kinase signal-integrating kinases Mnk1 and Mnk2. Although the role of eIF4E phosphorylation in mammalian cells has remained elusive, recent work in Drosophila has established that it is required for growth and development (Lachance, 2002). This study demonstrates that a previously identified Drosophila kinase called Lk6 is the functional homolog of mammalian Mnk kinases. lk6 loss-of-function alleles were generated and it was found that eIF4E phosphorylation is dramatically reduced in lk6 mutants. Importantly, lk6 mutants exhibit reduced viability, slower development, and reduced adult size, demonstrating that Lk6 function is required for organismal growth. Moreover, it is shown that uniform lk6 expression rescues the lethality of eIF4E hypomorphic mutants in an eIF4E phosphorylation site-dependent manner and that the two proteins participate in a common complex in Drosophila S2 cells, confirming the functional link between Lk6 and eIF4E. This work demonstrates that Lk6 exerts a tight control on eIF4E phosphorylation and is necessary for normal growth and development (Arquier, 2005).

To investigate the regulation of eIF4E phosphorylation in the physiological context of a developing organism, the Drosophila genome was scanned for kinases related to the vertebrate Mnk proteins. The best match corresponded to the sequence of a predicted kinase encoded by the lk6 gene. The Lk6 kinase was previously identified as a putative centrosomal, microtubules-associated protein, although a clear centrosomal function for Lk6 has not been established (Kidd, 1997). Alignment of the Lk6 and human Mnk1/2 protein sequences shows a high level of similarity in the kinase domain (65%–76%), as well as the conservation of specific features of the Mnk kinases, such as the presence of three regulatory threonines, an N-terminal stretch of basic amino acids involved in eIF4G binding in vertebrate Mnks, and a conserved MAPK binding sequence. It is therefore proposed that Lk6 is the Drosophila Mnk ortholog (Arquier, 2005).

To investigate the function of lk6, the P element EP3333, inserted in the first intron of the gene, was mobilized. Two imprecise excisions were obtained that delete part of the lk6 locus and thus may constitute lk6 loss-of-function alleles. lk6¹ contains a 3 kb deletion, which removes an alternative 5′ exon but leaves intact the rest of the coding sequence, allowing the production of lk6-A but not lk6-B transcripts. Homozygous lk6¹ mutant flies show reduced viability (20% lethality) and are fertile. Adult lk6¹ flies emerge with a 1–2 day developmental delay and a reduction of mass of 13% in males and 20% in females compared to the wild-type. lk6² deletes all lk6 exons downstream of the EP3333 insertion site, as well as sequences 5′ of the neighboring gene CG6923. Homozygous lk6² animals die as young second-instar larvae with dramatic growth defects. To exclude the possibility that some of these defects may result from the deletion of functional sequences in CG6923, the lk6¹/lk6² heteroallelic combination, which shows a more severe phenotype than lk6¹ homozygotes, was examined. lk6¹/lk6² flies have reduced viability (45% lethality) and a 20% reduction in mass in adult males and 25% in females as compared to heterozygous controls. lk6¹/lk6² mutant larvae present growth defects, as manifested in endoreplicating tissues such as the fat body and the salivary glands (30% size reduction for fat body cells). When measured in the adult wing, growth reduction (12%) appears primarily caused by a defect in cell number with barely any effect on cell size, indicative of a balanced reduction of cell growth and cell proliferation. The stronger phenotype of lk6¹/lk6² compared with lk6¹ homozygotes confirms that lk6¹ is a partial loss-of-function allele. Ubiquitous da-GAL4-driven expression of an lk6^wt construct rescued the lethality, the delayed development, and most of the size reduction associated with lk6¹/lk6² combination, confirming that mutant animals are indeed deficient for lk6 function (Arquier, 2005).

lk6¹/lk6² mutants present a mass reduction, development delay, and lethality comparable to eIF4E mutants expressing a nonphosphorylatable form of eIF4E (eIF4E^Ser251Ala) from a minigene (Lachance, 2002). This and the similarity between Lk6 and mammalian Mnks suggested that lk6 mutants might be deficient in eIF4E phosphorylation. To test this possibility, the level of eIF4E phosphorylation was compared in wild-type and lk6¹/lk6² mutant ovaries after metabolic labeling with ³²P-orthophosphate of eIF4E. eIF4E immunoprecipitated from mutant ovaries incorporates only 10% of the [³²P]-orthophosphate found in the control, revealing a 90% reduction of eIF4E kinase activity in the mutant. Thus, Lk6 kinase is required for physiological levels of eIF4E phosphorylation in flies, suggesting that Lk6 acts as an eIF4E kinase in vivo (Arquier, 2005).

To further confirm the link between eIF4E and Lk6, possible genetic interplay between the two genes was examined during development. eIF4E mutants arrest larval growth at various stages depending on allele strength and never reach late larval stages or pupariate. eIF4E^67Af mutants exhibit development arrest at the first larval instar, whereas homozygous eIF4E^Scim-a animals survive to the third instar (Lachance, 2002). Although uniform lk6 expression causes no growth phenotype on its own, it suppressed the larval lethality of an eIF4E^Scim-a/eIF4E^67Af hypomorphic combination. Interestingly, expression of a modified Lk6 protein (Lk6^T424D), mimicking a constitutively active version of mammalian Mnk1 (T332D) (Waskiewicz, 1999), better rescued growth in eIF4E mutant larvae, allowing development to small pharate adults. Remarkably, expression of lk6^T424D in the weaker eIF4E^Scim-a/eIF4E^Scim-a homozygous combination led to the emergence of viable, albeit small, adults. These results indicate that the major growth and developmental defects resulting from reduced eIF4E function are efficiently compensated by increased Lk6 kinase activity (Arquier, 2005).

If lk6^T424D expression rescues eIF4E mutants through the ability of the kinase to interact with phosphorylatable eIF4E, it should not further rescue an eIF4E mutant expressing a nonphosphorylatable form of eIF4E (eIF4E^Ser251Ala). This hypothesis was tested by expressing a UAS-eIF4E^Ser251Ala construct under the control of the ubiquitous daughterless-Gal4 driver in an eIF4E^Scim-a/eIF4E^67Af background. In these conditions, eIF4E^Ser251Ala expression only weakly rescues the larval lethality of the mutant, whereas eIF4E^wt expression provides a complete rescue. Coexpression of lk6^T424D with eIF4E^Ser251Ala did not provide a better rescue, indicating that Lk6 cannot act through nonphosphorylatable eIF4E^Ser251Ala. Overall, these data demonstrate that Lk6 positively controls eIF4E function through its phosphorylation and is required for growth and development (Arquier, 2005).

In support of this conclusion, it was found that, once expressed in Drosophila S2 cells, tagged Lk6 coimmunoprecipitated endogenous eIF4E. Reciprocally, tagged eIF4E also coimmunoprecipitated endogenous Lk6. The presence of excess eIF4E correlated with accumulation of endogenous Lk6, suggesting that Lk6 could be stabilized upon association with eIF4E-containing complexes. These results demonstrate that Lk6 and eIF4E are partners in a common protein complex in insect cells. In this respect, it is worth noting that Lk6 has a conserved eIF4G binding motif analogous to mammalian Mnks, possibly enabling it to bind to the initiation complex (Arquier, 2005).

This work establishes that loss of lk6 function compromises eIF4E phosphorylation and normal growth and development in flies. lk6 expression efficiently compensates for a reduction in eIF4E function, and the two proteins are part of a common biochemical complex in vivo. Overall, this supports the notion that Lk6 controls organismal growth through eIF4E phosphorylation. lk6 mutant flies have reduced wing size caused by reduced cell number. This phenotype is slightly different from what is observed in eIF4E^Ser251Ala-rescued eIF4E mutants, in which growth reduction in the adult eye results mostly from reduced ommatidial size and only slightly from a reduction in their number (Lachance, 2002). This difference could be explained if Lk6 acts on other targets in addition to eIF4E (Arquier, 2005).

In mammalian cells, Mnks are phosphorylated by Erk and p38 kinases on two conserved Threonine residues in their catalytic domain (Fukunaga, 1997; Waskiewicz, 1997). Whereas Mnk2a exhibits a nonregulated high basal activity, phosphorylation of Mnk1 by MAP kinases greatly enhances its eIF4E kinase activity (for review see Scheper, 2002b). Interestingly, the Erk/p38-targeted residues, as well as the MAPK binding domain present in Mnks, are conserved in Lk6. This, as well as the fact that Lk6 was found in an overexpression screen for modifiers of the ras-signaling pathway in the Drosophila eye (Huang, 2000), suggests that, as in the case of Mnks, Lk6 activity is linked to the ras/MAP kinase cascade in vivo (Arquier, 2005).

Although phosphorylation of eIF4E has been consistently associated with activation of protein synthesis, studies in mammalian tissue culture cells have yielded contradictory results concerning the functional significance of eIF4E phosphorylation during translation initiation. In particular, recent biophysical data established that phosphorylation of eIF4E reduces its affinity for capped mRNA (Scheper, 2002a), suggesting a model in which eIF4E phosphorylation by Mnk kinases triggers its release from the cap structure, therefore allowing recycling and further recruitment of a new initiation complex on the cap. Facilitation of translation initiation through phosphorylation of eIF4E is in agreement with the demonstration of a positive role for Lk6 activity in controlling cell and tissue growth. According to this model, forced eIF4E phosphorylation might cause constitutive destabilization of cap complex and thus be detrimental to translation initiation, as already suggested in mammalian cells (Knauf, 2001). Indeed, in the course of the current experiments, it was observed that strong Lk6 overexpression in the eye disc leads to subtle growth impairments. This suggests that a precise control of eIF4E phosphorylation is necessary for optimal translation and growth machinery function in vivo (Arquier, 2005).

Recent genetic analysis of double Mnk1/Mnk2 knockout mice has revealed that both kinases are dispensable for growth and development (Ueda, 2004). This difference with results in the fly could be because of redundancies or compensatory mechanisms taking place in the regulatory circuitry of mammals. The Drosophila system might thus provide a simpler version of an integrated developing model, allowing important cell regulatory mechanisms to be uncovered (Arquier, 2005).

Diet-dependent effects of the Drosophila Mnk1/Mnk2 homolog Lk6 on growth via eIF4E

The control of cellular growth is tightly linked to the regulation of protein synthesis. A key function in translation initiation is fulfilled by the 5' cap binding eukaryotic initiation factor 4E (eIF4E), and dysregulation of eIF4E is associated with malignant transformation and tumorigenesis. In mammals, the activity of eIF4E is modulated by phosphorylation at Ser209 by mitogen-activated protein kinases (MAPK)-interacting kinases 1 and 2 (Mnk1 and Mnk2), which themselves are activated by ERK and p38 MAPK in response to mitogens, cytokines or cellular stress. Whether phosphorylation of eIF4E at Ser209 exerts a positive or inhibitory effect on translation efficiency has remained controversial. This study provides a genetic characterization of the Drosophila homolog of Mnk1/2, Lk6. Lk6 function is dispensable under a high protein diet, consistent with the recent finding that mice lacking both Mnk1 and Mnk2 are not growth-impaired (Ueda, 2004). Interestingly, loss of Lk6 function causes a significant growth reduction when the amino acid content in the diet is reduced. Lk6 expression has also shown to be upregulated upon starvation during larval development (Zinke, 2002). Overexpression of Lk6 also results in growth inhibition in an eIF4E-dependent manner. A model of eIF4E regulation is provided that may reconcile the contradictory findings with regard to the role of phosphorylation by Mnk1/2 (Reiling, 2005).

Evidence is provided that Lk6 exerts its function via phosphorylation of eIF4E because the effects of Lk6 overexpression are strictly dependent on the presence of Ser251 in eIF4E. This conclusion is strongly supported by the finding that eIF4E phosphorylation is diminished in ovaries of Lk6 mutant flies. Therefore, flies lacking Lk6 function can be expected to display the same phenotype as eIF4E mutant flies rescued by a P{eIF4E^Ser251Ala transgene. However, the rescued eIF4E mutants grow to a smaller size even under standard culture conditions. Although they contain significantly fewer cells, the size reduction is predominantly caused by smaller cells. In contrast, the loss of Lk6 function primarily affects cell number. Whether these discrepancies reflect a qualitative difference between eIF4E mutant flies rescued by a P{eIF4E^Ser251Ala} transgene and Lk6 mutants is currently unknown (Reiling, 2005).

It is speculated that the net result of Lk6/Mnk activity (i.e., whether translation is inhibited or promoted) is not determined by the absolute levels of Lk6/Mnk, but rather by the ratio of activated Lk6/Mnk and free eIF4E (i.e., not bound by 4E-BPs), the limiting factor for translation initiation. Under standard culture conditions (high protein), a larger fraction of eIF4E assembles into functional eIF4F complexes because of high TOR activity, thereby promoting translation. Under reduced conditions (e.g., 30% yeast), TOR pathway activity is lowered, and, thus, more 4E-BP binds and inhibits eIF4E, which dampens the rate of translation. It is likely that the predominant mechanism of eIF4E regulation is achieved by TOR/4E-BP activity, and that the phosphorylation of eIF4E by Lk6/Mnk imposes a translational fine-tuning that becomes rate limiting only under adverse food conditions. Lacking Lk6 function in addition to diminished eIF4E availability impinges on translation efficiency, which results in the observed body size reduction (Reiling, 2005).

Alternatively, high TOR activity caused by a diet rich in amino acids could enable the activation of another (unidentified) eIF4E kinase that acts redundantly to Lk6/Mnk. However, this is rather unlikely because mice lacking Mnk1 and Mnk2 do not show any residual eIF4E-Ser209 phosphorylation, strongly arguing against an uncharacterized eIF4E kinase (Reiling, 2005).

Overexpression of Lk6 under standard food conditions consistently resulted in a suppression of growth. Furthermore, another EP insertion in the Lk6 locus (EP3344), which promotes lower expression levels as compared to EPLk6, yielded qualitatively similar but milder phenotypes, suggesting that the dosage of Lk6 expression is important for its ability to regulate growth. Concentration-dependent effects of Mnk1 have also been described by Knauf (2001), who suggested a negative role of Mnk1/2 for cap-dependent translation. It is conceivable that overexpressed Lk6 exerts a dominant-negative effect on translation efficiency by reducing the affinity of phosphorylated eIF4E for capped mRNA, leading to a precocious disassembly of the eIF4F complex (Reiling, 2005).

Reducing the amino acid supply abolished the negative effects of Lk6 overexpression on growth, suggesting that the activity of Lk6 is also regulated in response to nutrients. The mechanism for this additional layer of regulation is unknown but is likely to involve phosphorylation by the upstream kinases ERK and/or p38. Consistently, the p38 homolog in fission yeast, Sty1/Spc1, is regulated in response to nutrient limitation and osmotic stress (Reiling, 2005).

The effects of Lk6 activity are therefore context dependent: They lead either to growth stimulation or growth inhibition. It is proposed that (1) the timing, (2) the amount of eIF4E phosphorylation during 48S complex assembly, and (3) nutrient (amino acid) availability are critical parameters for the modulation of growth by Lk6 (Reiling, 2005).

The results underscore the importance of the diet composition for growth studies. In fact, under the standard food conditions used, the Lk6 mutant growth phenotype would have escaped detection, demonstrating that different food compositions are likely to result in qualitatively different outcomes of otherwise identical experiments. Researchers in the growth field, ourselves included, have not paid sufficient attention to what their flies actually eat. Therefore, it is proposed that Drosophila geneticists should always describe the composition of the fly food when reporting on growth-related experiments. Ideally, a global standard fly medium should be defined (Reiling, 2005).

The relationship between nutrition and growth has also been appreciated by others. It has been estimated that 14%–20% of all cancer deaths in the US are attributable to overweight and obesity. Therefore, understanding the role of diet in cancer development will represent a crucial task in the future (Reiling, 2005).

Nutritional control of protein biosynthetic capacity by insulin via Myc in Drosophila

Animals use the insulin/TOR signaling pathway to mediate their response to fluctuations in nutrient availability. Energy and amino acids are monitored at the single-cell level via the TOR branch of the pathway and systemically via insulin signaling to regulate cellular growth and metabolism. Using a combination of genetics, expression profiling, and chromatin immunoprecipitation, this study examined nutritional control of gene expression and identified the transcription factor Myc as an important mediator of TOR-dependent regulation of ribosome biogenesis. myc was also identified as a direct target of FOXO, and genetic evidence is provided that Myc has a key role in mediating the effects of TOR and FOXO on growth and metabolism. FOXO and TOR also converge to regulate protein synthesis, acting via 4E-BP and Lk6, regulators of the translation factor eIF4E. This study uncovers a network of convergent regulation of protein biosynthesis by the FOXO and TOR branches of the nutrient-sensing pathway (Teleman, 2008).

The global transcriptional analysis reported in this study has revealed a surprising degree of interconnectedness between the two branches of the nutrient-sensing pathway. Insulin, acting through PI3K and Akt, feeds into the FOXO and TORC1 branches of the pathway, whereas energy levels (AMP/ATP) and amino acids act directly on the TORC1 branch. How are these inputs integrated to maintain energy balance? It was previously known that 4E-BP is transcriptionally regulated by FOXO and posttranslationally regulated by TOR. This study has identified the protein kinase Lk6 as a second direct FOXO target. Thus, there appear to be two parallel, independent mechanisms by which the TOR and FOXO branches of the insulin signaling pathway converge to regulate eIF4E activity and hence cellular protein translation. This 'belt and suspenders' approach to translational control might be important to make the system robust (Teleman, 2008).

A key finding of this study is the identification of Myc as a point of convergent regulation by the FOXO and TOR branches of the pathway. myc mRNA levels are controlled by FOXO in a tissue-specific manner. In addition, Myc protein levels are dependent on TORC1. Why use two independent means to control Myc levels? Transcription alone would limit the speed with which the system can respond to changing nutritional conditions. This might be detrimental, particularly as conditions worsen. Regulation of Myc activity by TORC1 permits a rapid response to changes in energy levels or amino acid availability and could serve to fine tune the nutritional response in the cell by controlling translational outputs. This parallels the situation with 4E-BP, albeit with a slightly different logic. Reduced insulin signaling allows FOXO to enter the nucleus and increase 4E-BP expression and at the same time alleviates TORC1-mediated inhibition of the existing pool of 4E-BP. A subsequent increase in energy or amino acid levels would permit rapid reinhibition of 4E-BP and thus allow a flexible response during the time needed for the pool of protein elevated in response to reduced insulin levels to decay (Teleman, 2008).

In yeast, TORC1 is known to regulate ribosome biogenesis through different nuclear RNA polymerases. It has been shown that yeast TORC1 can bind DNA directly at the 35S rDNA promoter and activate Pol I-mediated transcription in a rapamycin-sensitive manner. Moreover, yeast TORC1 is known regulate Pol II-dependent RP gene expression by controlling the nuclear localization of the transcription factor SFP1 and CRF1, a corepressor of the forkhead transcription factor FHL1. In Drosophila, TORC1 has recently been reported to regulate a set of protein-coding genes involved in ribosome assembly. This study has identified Myc as the missing link mediating TORC1-dependent regulation of this set of genes. Indeed, the fact that more than 90% of TORC1-activated genes contain E boxes suggests that Myc might be the main mediator of this transcriptional program. This connection suggests that expression of Myc targets as a whole should be responsive to nutrient conditions. Indeed, this study found that 33% of direct Myc targets -- defined as genes reported to be bound by Myc when assayed by DNA adenine methyltransferase ID (DamID) in Kc cells and to be regulated by myc overexpression in larvae -- are downregulated upon nutrient deprivation. This is a significant enrichment of 4-fold relative to all genes in the genome, despite the comparison being based on correlating data from different tissue types (Teleman, 2008).

It seems reasonable that cellular translation rates need to be dampened if the TOR branch of the pathway senses low amino acid levels. As ribosome biogenesis is energetically expensive, it may be advantageous to link ribosome biogenesis and translational control via TORC1. This dual regulation is well reflected in tissue growth, since this study observed that Myc, the regulator of ribosome biogenesis, is essential for tissue growth driven by the TOR pathway but not sufficient to drive growth in the absence of TOR activity. The FOXO branch of the pathway senses reduced insulin or mitogen levels. FOXO is also highly responsive to oxidative and other stresses and would integrate this information into the cellular control of translation. The data support the notion of a network in which TOR and FOXO regulate protein biosynthesis by converging on Myc to regulate ribosome biogenesis and on eIF4E activity via 4E-BP and Lk6 to regulate translation initiation (Teleman, 2008).

The work presented in this study complements a previous study in which larvae were either starved completely or starved for amino acids only, while having a supply of energy in the form of sugar. A significant and positive correlation (~0.4) indicates general agreement between the two data sets, but they differ in two ways. The current goal was to explore the regulatory network by which insulin controls cellular transcription. Individual tissues were isolated rather than assaying the whole animal. Genes found to be regulated in a previous but not in the current assays may be regulated in tissues other than muscle or adipose tissue. Conversely, genes identified only by the current study might be regulated oppositely in different tissues or might only be regulated in a subset of tissues and so be missed in a whole-animal analysis.

Is Myc also involved in nutritional signaling networks in mammals? No similar rapid downregulation of c-myc was seen in response to rapamycin in human cell lines, suggesting that the mechanism by which TOR signaling controls gene expression may differ between phyla. This is further supported by the fact that the sets of genes reported to be rapamycin regulated also appear to be largely distinct in Drosophila and mammalian cells, with the caveat that different cell types were used in the two analyses. Although the mechanism does not appear to be identical in mammals, there are several suggestions in the literature of a connection between c-Myc and nutritional signaling. For example, dMyc and c-Myc share the ability to regulate ribosome biogenesis, although the specific target genes through which they do so are different. There is also evidence that mammalian c-myc expression in liver is regulated by nutrition and that transgenic expression of c-myc in liver affects metabolism, i.e., glucose uptake and gluconeogenesis. Furthermore, it has been reported that FOXO3 represses Myc activity in colon cancer cells by inducing members of the Mad/Mxi family, which are known to antagonize Myc. The current data suggest that Max and Mnt are not transcriptionally regulated by insulin or FOXO in Drosophila, whereas myc is. This is similar to what has been reported in murine lymphoid cells, in which c-myc expression is regulated by the FOXO homolog FKHRL1. These parallels between the fly and mammalian systems suggest a broader connection between insulin signaling and activity of the Myc/Mnt/Max network. Although some features may be different in the two systems, the similarities merit further investigation (Teleman, 2008).

Finally, this work has revealed a surprising amount of tissue specificity in the transcriptional response to insulin signaling. Roughly half of the genes regulated by insulin in adipose tissue or in muscle were not significantly regulated in the other tissue. Furthermore, 155 genes were differentially regulated in the two tissues (i.e., upregulated in one tissue and downregulated in the other). This likely reflects the roles of the different tissues in the organism's response to nutrient deprivation. Further work will elucidate the underlying molecular mechanisms (Teleman, 2008).

The extracellular-regulated kinase effector Lk6 is required for Glutamate receptor localization at the Drosophila neuromuscular junction

The proper localization and synthesis of postsynaptic glutamate receptors are essential for synaptic plasticity. Synaptic translation initiation is thought to occur via the target of rapamycin (TOR) and mitogen-activated protein kinase signal-integrating kinase (Mnk) signaling pathways, which is downstream of extracellular-regulated kinase (ERK). This study used the model glutamatergic synapse, the Drosophila neuromuscular junction, to better understand the roles of the Mnk and TOR signaling pathways in synapse development. These synapses contain non-NMDA receptors that are most similar to AMPA receptors. The data show that Lk6, the Drosophila homolog of Mnk1 and Mnk2, is required in either presynaptic neurons or postsynaptic muscle for the proper localization of the GluRIIA glutamate receptor subunit. Lk6 may signal through eukaryotic initiation factor (eIF) 4E to regulate the synaptic levels of GluRIIA as either interfering with eIF4E binding to eIF4G or expression of a nonphosphorylatable isoform of eIF4E resulted in a significant reduction in GluRIIA at the synapse. It was also found that Lk6 and TOR may independently regulate synaptic levels of GluRIIA. (Hussein, 2016).

This study is the first to provide information on the properties and regulation of the Drosophila protein kinase LK6. Its catalytic domain is strikingly similar to those of mammalian Mnks; similar to them, in mammalian cells LK6 can bind to ERK, can be activated by ERK signalling and can phosphorylate eIF4E. This occurs at the physiological site, Ser²⁰⁹. The MAPK-binding motif of LK6 is of the type previously shown to bind ERK but not p38 MAPK. Consistent with this, when expressed in mammalian cells, LK6 is not activated by stimuli that turn on p38 MAPK (Hussein, 2016).

It is more challenging to perform similar experiments in Drosophila cells owing to the difficulty in transfecting, e.g. S2 cells with high efficiency. However, importantly, this study shows that LK6 also interacts with the ERK homologue Rolled, but not with the Drosophila p38 homologue. The results, furthermore, show that LK6 is activated by Phorbol myristate acetate (PMA), but not by arsenite, which activates p38 MAPK. The regulatory properties of LK6 thus appear to be similar in mammalian and Drosophila cells, indicating that the specificity of the MAPK-interaction motifs is probably similar in both mammals and Diptera. Similar to Mnk1 and Mnk2a, LK6 is primarily, if not exclusively, cytoplasmic. It does contain a basic region of the type that, in Mnk1 and Mnk2, can bind to the nuclear shuttling protein importin-α. It therefore seems probable that either (1) it contains an NES, which ensures its efficient re-export from the nucleus, or (2) the basic region is not accessible to importin-α. The lack of effect of LMB on the localization of LK6 rules out the operation of a CRM1-type NES of the kind found in Mnk1, although the very long C-terminal extension of LK6 might contain an LMB-insensitive NES (Hussein, 2016).

By analogy with the Mnks, it is probable that the N-terminal polybasic region of LK6 mediates its binding to eIF4G and could also interact with importin-α. Given that full-length LK6 shows less efficient binding to eIF4G when compared with Mnk1, it also seems possible that it binds importin-α less efficiently, which may contribute to the finding that LK6 is cytoplasmic. It has been shown previously that even the much shorter C-terminus of Mnk2a impedes access to the N-terminal basic region in that protein, so it is entirely possible that the much larger C-terminal part of LK6 has a similar effect. This could explain why the fragment of LK6 that lacks the C-terminus bound better to eIF4G than did the full-length protein. It may also be that the low degree of binding reflects the fact that the association of LK6 with the heterologous human protein was being studied, rather than with Drosophila eIF4G. Repeated attempts have been made to use the available antisera to examine the association of LK6 with eIF4G in S2 cells, but without success. Comparison of the polybasic region of LK6 with those of Mnk1 and Mnk2a (which do bind eIF4G and importin-α), and recent results for mutants with alterations in these features, do not reveal any difference that might obviously explain the decreased ability of LK6 to bind mammalian eIF4G. As argued above, the C-terminus of LK6 may also impair its activation by ERK, based on the observation that the catalytic domain is more effectively activated than a mutant of the full-length protein that also lacks the ERK-binding motif (Hussein, 2016).

The results support the idea that LK6 is a Drosophila eIF4E kinase. LK6 can phosphorylate eIF4E in vitro and its overexpression in cells leads to increased phosphorylation of endogenous eIF4E. Furthermore, the activation of LK6 by ERK signalling but not by p38 MAPK signalling correlates well with the observed behaviour of the phosphorylation of eIF4E in PMA- or arsenite-treated Drosophila cells, and the fact that LK6 is activated by stimuli that stimulate ERK but is not activated by stimuli that activate p38 MAPK, in HEK-293 cells. The ability of LK6 to bind eIF4G also supports the contention that it can act as an eIF4E kinase in vivo (Hussein, 2016).

The observation that phosphorylation of the endogenous eIF4E in S2 cells is increased by PMA but not by arsenite is consistent with the regulatory properties of LK6 and with the notion that LK6 may phosphorylate eIF4E in these cells. The fact that it is the only close homologue of the Mnks in the fruitfly genome is also consistent with this notion. Phosphorylation of eIF4E has previously been shown to play an important role in growth in this organism and in its normal development. The current data show that LK6 can phosphorylate Drosophila eIF4E in vitro, consistent with the idea that LK6 acts as an eIF4E kinase in this organism. The dsRNAi data that was obtained, which show that two different interfering dsRNAs directed against LK6 each markedly decrease eIF4E phosphorylation in S2 cells, offer strong support to the conclusion that LK6 acts as an eIF4E kinase in Drosophila. Unfortunately, the poor quality of the available anti-LK6 antisera prevented assessing whether the incomplete nature of the loss of phosphorylation of eIF4E reflects incomplete elimination of LK6 expression (Hussein, 2016).

Previous genetic studies have linked LK6 to Ras signalling in Drosophila. This agrees very well with the finding that LK6 is activated by ERK signalling, since ERK lies downstream of Ras. LK6 was first identified as interacting with microtubules and centrosomes. Overexpression of LK6 led to defects in microtubule organization, indicative of their increased stability. The connections between the phosphorylations of eIF4E and microtubules are not immediately obvious. However, it is entirely possible that LK6 has additional substrates that interact with microtubules or are components of centrosomes and their phosphorylation may be important in the regulation of, for example, mitosis. Numerous microtubule-associated proteins are indeed phosphorylated. Microtubules undergo massive reorganization during mitosis and this involves an array of phosphorylation events and protein kinases. It may therefore be relevant that LK6 is activated by mitogenic signalling (i.e. through ERK and thus Ras) (Hussein, 2016).

LK6 is regulated by ERK and phosphorylates the eukaryotic initiation factor eIF4E in vivo

In Drosophila cells, phosphorylation of eIF4E (eukaryotic initiation factor 4E) is required for growth and development. In Drosophila, LK6 is the closest homologue of mammalian Mnk1 and Mnk2 [MAPK (mitogen-activated protein kinase) signal-integrating kinases 1 and 2 respectively] that phosphorylate mammalian eIF4E. Mnk1 is activated by both mitogen- and stress-activated signalling pathways [ERK (extracellular-signal-regulated kinase) and p38 MAPK], whereas Mnk2 contains a MAPK-binding motif that is selective for ERKs. LK6 possesses a binding motif similar to that in Mnk2. The present study shows that LK6 can phosphorylate eIF4E at the physiological site. LK6 activity is increased by the ERK signalling pathway and not by the stress-activated p38 MAPK signalling pathway. Consistent with this, LK6 binds ERK in mammalian cells, and this requires an intact binding motif. LK6 can bind to eIF4G in mammalian cells, and expression of LK6 increases the phosphorylation of the endogenous eIF4E. In Drosophila S2 Schneider cells, LK6 binds the ERK homologue Rolled, but not the p38 MAPK homologue. LK6 phosphorylates Drosophila eIF4E in vitro. The phosphorylation of endogenous eIF4E in Drosophila cells is increased by activation of the ERK pathway but not by arsenite, an activator of p38 MAPK. RNA interference directed against LK6 significantly decreases eIF4E phosphorylation in Drosophila cells. These results show that LK6 binds to ERK and is activated by ERK signalling and it is responsible for phosphorylating eIF4E in Drosophila (Parra-Palau, 2005; full text of article).

p38 MAP Kinase regulates circadian rhythms in Drosophila

The large repertoire of circadian rhythms in diverse organisms depends on oscillating central clock genes, input pathways for entrainment, and output pathways for controlling rhythmic behaviors. Stress-activated p38 MAP Kinases (p38K), although sparsely investigated in this context, show circadian rhythmicity in mammalian brains and are considered part of the circadian output machinery in Neurospora. This study found that Drosophila p38Kb is expressed in clock neurons, and mutants in p38Kb either are arrhythmic or have a longer free-running periodicity, especially as they age. Paradoxically, similar phenotypes are observed through either transgenic inhibition or activation of p38Kb in clock neurons, suggesting a requirement for optimal p38Kb function for normal free-running circadian rhythms. This study also found that p38Kb genetically interacts with multiple downstream targets to regulate circadian locomotor rhythms. More specifically, p38Kb interacts with the period gene to regulate period length and the strength of rhythmicity. In addition, p38Kb was shown to suppress the arrhythmic behavior associated with inhibition of a second p38Kb target, the transcription factor Mef2. Finally, manipulating p38K signaling in free-running conditions was found to alter the expression of another downstream target, MNK/Lk6, which has been shown to cycle with the clock and to play a role in regulating circadian rhythms. These data suggest that p38Kb may affect circadian locomotor rhythms through the regulation of multiple downstream pathways (Vrailas-Mortimer, 2014).

Search PubMed for articles about Drosophila lk6

Arquier, N., Bourouis, M., Colombani, J. and Leopold, P. (2005). Drosophila Lk6 kinase controls phosphorylation of eukaryotic translation initiation factor 4E and promotes normal growth and development. Curr. Biol. 15(1): 19-23. PubMed ID; Online text

Fukunaga, R. and Hunter, T. (1997). MNK1, a new MAP kinase-activated protein kinase, isolated by a novel expression screening method for identifying protein kinase substrates. EMBO J. 16: 1921-1933. PubMed ID: 9155018

Huang, A. M. and Rubin, G. M. (2000). A misexpression screen identifies genes that can modulate RAS1 pathway signaling in Drosophila melanogaster. Genetics 156: 1219-1230. PubMed ID: 11063696

Hussein, N. A., Delaney, T. L., Tounsel, B. L. and Liebl, F. L. (2016). The extracellular-regulated kinase effector Lk6 is required for Glutamate receptor localization at the Drosophila neuromuscular junction. J Exp Neurosci 10: 77-91. PubMed ID: 27199570

Kidd, D. and Raff, J. W. (1997). LK6, a short lived protein kinase in Drosophila that can associate with microtubules and centrosomes. J. Cell Sci.110: 209-199044051

Knauf, U., Tschopp, C. and Gram, H. (2001). Negative regulation of protein translation by mitogen-activated protein kinase-interacting kinases 1 and 2. Mol. Cell. Biol. 21: 5500-5511. PubMed ID: 11463832

Lachance, P. E., et al. (2002). Phosphorylation of eukaryotic translation initiation factor 4E is critical for growth. Mol. Cell. Biol. 22: 1656-1663. PubMed ID: 11865045

Parra-Palau, J. L., Scheper, G. C., Harper, D. E. and Proud, C. G. (2005). The Drosophila protein kinase LK6 is regulated by ERK and phosphorylates the eukaryotic initiation factor eIF4E in vivo. Biochem. J. 385(Pt 3): 695-702. PubMed ID: 15487973

Reiling, J. H., Doepfner, K. T., Hafen, E. and Stocker, H. (2005). Diet-dependent effects of the Drosophila Mnk1/Mnk2 homolog Lk6 on growth via eIF4E. Curr. Biol. 15(1): 24-30. PubMed ID: 15649360

Scheper, G. C., et al. (2002a). Phosphorylation of eukaryotic initiation factor 4E markedly reduces its affinity for capped mRNA. J. Biol. Chem. 277: 3303-3309. PubMed ID: 11723111

Scheper, G. C. and Proud, C. G. (2002b). Does phosphorylation of the cap-binding protein eIF4E play a role in translation initiation?. Eur. J. Biochem. 269: 5350-5359. PubMed ID: 12423333

Teleman, A. A., Hietakangas, V., Sayadian, A. C. and Cohen, S. M. (2008). Nutritional control of protein biosynthetic capacity by insulin via Myc in Drosophila. Cell Metab 7: 21-32. PubMed ID: 18177722

Ueda, T., et al. (2004). Mnk2 and Mnk1 are essential for constitutive and inducible phosphorylation of Eukaryotic Initiation Factor 4E but not for cell growth or development. Mol. Cell. Biol. 24: 6539-6549. PubMed ID: 15254222

Vrailas-Mortimer, A. D., Ryan, S. M., Avey, M. J., Mortimer, N. T., Dowse, H. and Sanyal, S. (2014). p38 MAP Kinase regulates circadian rhythms in Drosophila. J Biol Rhythms [Epub ahead of print]. PubMed ID: 25403440

Waskiewicz, A. J., et al. (1997). Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2. EMBO J. 16: 1909-1920. PubMed ID: 9155017

Waskiewicz, A. J. et al. (1999). Phosphorylation of the cap-binding protein eukaryotic translation initiation factor 4E by protein kinase Mnk1 in vivo. Mol. Cell. Biol. 19: 1871-1880. PubMed ID: 10022874

Zinke, I., et al. (2002). Nutrient control of gene expression in Drosophila: Microarray analysis of starvation and sugar-dependent response. EMBO J. 21: 6162-6173. PubMed ID: 12426388

date revised: 21 November 2016

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