InteractiveFly: GeneBrief

Lk6: Biological Overview | References

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).

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).

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 citation; 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 citation: 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 citation: 11063696

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 citation: 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 citation: 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 citation: 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 citation: 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 citation: 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 citation: 12423333

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 citation: 15254222

Waskiewicz, A. J., et al. (1997). Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2. EMBO J. 16: 1909-1920. PubMed citation: 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 citation: 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 citation: 12426388

date revised: 25 April 2008

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