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Lipid phosphate phosphatases dimerise, but this interaction is not required for in vivo activity.

Burnett C, Makridou P, Hewlett L, Howard K - BMC Biochem. (2004)

Bottom Line: Furthermore, Wunen does not form dimers with its closely related counterpart Wunen-2.Since neither dimerisation nor the C-terminus seem to be involved in substrate recognition, they may instead confer structural or functional stability through dimerisation.The results indicate that the associations we see are highly specific and occur only between monomers of the same protein.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, MRC Laboratory for Molecular Cell Biology, University College London, Gower St, London WC1E 6BT, UK. c.burnett@ucl.ac.uk

ABSTRACT

Background: Lipid phosphate phosphatases (LPPs) are integral membrane proteins believed to dephosphorylate bioactive lipid messengers, so modifying or attenuating their activities. Wunen, a Drosophila LPP homologue, has been shown to play a pivotal role in primordial germ cell (PGC) migration and survival during embryogenesis. It has been hypothesised that LPPs may form oligomeric complexes, and may even function as hexamers. We were interested in exploring this possibility, to confirm whether LPPs can oligomerise, and if they do, whether oligomerisation is required for either in vitro or in vivo activity.

Results: We present evidence that Wunen dimerises, that these associations require the last thirty-five C-terminal amino-acids and depend upon the presence of an intact catalytic site. Expression of a truncated, monomeric form of Wunen in Drosophila embryos results in perturbation of germ cell migration and germ cell loss, as observed for full-length Wunen. We also observed that murine LPP-1 and human LPP-3 can also form associations, but do not form interactions with Wunen or each other. Furthermore, Wunen does not form dimers with its closely related counterpart Wunen-2. Finally we discovered that addition of a trimeric myc tag to the C-terminus of Wunen does not prevent dimerisation or in vitro activity, but does prevent activity in vivo.

Conclusion: LPPs do form complexes, but these do not seem to be specifically required for activity either in vitro or in vivo. Since neither dimerisation nor the C-terminus seem to be involved in substrate recognition, they may instead confer structural or functional stability through dimerisation. The results indicate that the associations we see are highly specific and occur only between monomers of the same protein.

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Ectopic expression Ectopic expression. Embryos a-c are viewed laterally with the posterior pole to the right, embryos d-i are viewed dorsally. Expression of WunD2GFP (a) at stage 10 has a similar phenotype to WunGFP (b) with an early and dramatic loss of PGCs compared to wild-type (c). By the end of embryogenesis, neither WunD2GFP (d) nor WunGFP (e) expressing embryos have formed gonads, as opposed to a wild-type embryo at the same stage (f). Expression of WunD:248>TGFP (g), WunD2M3 (h) or WunM3 (i) gives no discernable phenotype with all embryos forming two distinct gonads. Wild-type embryos have not been stained with anti-GFP and consequently show no blue staining.
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Figure 3: Ectopic expression Ectopic expression. Embryos a-c are viewed laterally with the posterior pole to the right, embryos d-i are viewed dorsally. Expression of WunD2GFP (a) at stage 10 has a similar phenotype to WunGFP (b) with an early and dramatic loss of PGCs compared to wild-type (c). By the end of embryogenesis, neither WunD2GFP (d) nor WunGFP (e) expressing embryos have formed gonads, as opposed to a wild-type embryo at the same stage (f). Expression of WunD:248>TGFP (g), WunD2M3 (h) or WunM3 (i) gives no discernable phenotype with all embryos forming two distinct gonads. Wild-type embryos have not been stained with anti-GFP and consequently show no blue staining.

Mentions: LPPs active in this assay, however, will not necessarily function in vivo. To examine the ability of the truncated, monomeric form to act on the PGC specific survival factor in Drosophila embryos, we generated transgenic flies with WunD2GFP and WunD2M3 and crossed them to the mesoderm driver Twist-GAL4 [12]. Ectopic expression of WunD2GFP gave a similar phenotype to ectopic expression of full-length Wun, with scattered germ cells, a failure to form functional gonads, and a marked reduction in germ cell number (Fig. 3a,3b,3c,3d,3e,3f). This indicates that neither dimerisation nor the C-terminal thirty-five amino-acids are required for activity in vivo. Surprisingly, expression of WunD2M3, which has activity in the LPA assay, had no affect on PGC migration or survival (Fig. 3h). We mis-expressed WunM3 in vivo and found that this too had a total loss of function, despite high levels of protein in the mesoderm and activity in the in vitro LPA assay (Fig. 3i). This indicates that in vivo, this myc tag prevents the function of a previously active protein and cannot be relied on as a passive tag. These results show that catalytic activity as assayed biochemically, is not a true reflection of activity as assayed in vivo.


Lipid phosphate phosphatases dimerise, but this interaction is not required for in vivo activity.

Burnett C, Makridou P, Hewlett L, Howard K - BMC Biochem. (2004)

Ectopic expression Ectopic expression. Embryos a-c are viewed laterally with the posterior pole to the right, embryos d-i are viewed dorsally. Expression of WunD2GFP (a) at stage 10 has a similar phenotype to WunGFP (b) with an early and dramatic loss of PGCs compared to wild-type (c). By the end of embryogenesis, neither WunD2GFP (d) nor WunGFP (e) expressing embryos have formed gonads, as opposed to a wild-type embryo at the same stage (f). Expression of WunD:248>TGFP (g), WunD2M3 (h) or WunM3 (i) gives no discernable phenotype with all embryos forming two distinct gonads. Wild-type embryos have not been stained with anti-GFP and consequently show no blue staining.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC319698&req=5

Figure 3: Ectopic expression Ectopic expression. Embryos a-c are viewed laterally with the posterior pole to the right, embryos d-i are viewed dorsally. Expression of WunD2GFP (a) at stage 10 has a similar phenotype to WunGFP (b) with an early and dramatic loss of PGCs compared to wild-type (c). By the end of embryogenesis, neither WunD2GFP (d) nor WunGFP (e) expressing embryos have formed gonads, as opposed to a wild-type embryo at the same stage (f). Expression of WunD:248>TGFP (g), WunD2M3 (h) or WunM3 (i) gives no discernable phenotype with all embryos forming two distinct gonads. Wild-type embryos have not been stained with anti-GFP and consequently show no blue staining.
Mentions: LPPs active in this assay, however, will not necessarily function in vivo. To examine the ability of the truncated, monomeric form to act on the PGC specific survival factor in Drosophila embryos, we generated transgenic flies with WunD2GFP and WunD2M3 and crossed them to the mesoderm driver Twist-GAL4 [12]. Ectopic expression of WunD2GFP gave a similar phenotype to ectopic expression of full-length Wun, with scattered germ cells, a failure to form functional gonads, and a marked reduction in germ cell number (Fig. 3a,3b,3c,3d,3e,3f). This indicates that neither dimerisation nor the C-terminal thirty-five amino-acids are required for activity in vivo. Surprisingly, expression of WunD2M3, which has activity in the LPA assay, had no affect on PGC migration or survival (Fig. 3h). We mis-expressed WunM3 in vivo and found that this too had a total loss of function, despite high levels of protein in the mesoderm and activity in the in vitro LPA assay (Fig. 3i). This indicates that in vivo, this myc tag prevents the function of a previously active protein and cannot be relied on as a passive tag. These results show that catalytic activity as assayed biochemically, is not a true reflection of activity as assayed in vivo.

Bottom Line: Furthermore, Wunen does not form dimers with its closely related counterpart Wunen-2.Since neither dimerisation nor the C-terminus seem to be involved in substrate recognition, they may instead confer structural or functional stability through dimerisation.The results indicate that the associations we see are highly specific and occur only between monomers of the same protein.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, MRC Laboratory for Molecular Cell Biology, University College London, Gower St, London WC1E 6BT, UK. c.burnett@ucl.ac.uk

ABSTRACT

Background: Lipid phosphate phosphatases (LPPs) are integral membrane proteins believed to dephosphorylate bioactive lipid messengers, so modifying or attenuating their activities. Wunen, a Drosophila LPP homologue, has been shown to play a pivotal role in primordial germ cell (PGC) migration and survival during embryogenesis. It has been hypothesised that LPPs may form oligomeric complexes, and may even function as hexamers. We were interested in exploring this possibility, to confirm whether LPPs can oligomerise, and if they do, whether oligomerisation is required for either in vitro or in vivo activity.

Results: We present evidence that Wunen dimerises, that these associations require the last thirty-five C-terminal amino-acids and depend upon the presence of an intact catalytic site. Expression of a truncated, monomeric form of Wunen in Drosophila embryos results in perturbation of germ cell migration and germ cell loss, as observed for full-length Wunen. We also observed that murine LPP-1 and human LPP-3 can also form associations, but do not form interactions with Wunen or each other. Furthermore, Wunen does not form dimers with its closely related counterpart Wunen-2. Finally we discovered that addition of a trimeric myc tag to the C-terminus of Wunen does not prevent dimerisation or in vitro activity, but does prevent activity in vivo.

Conclusion: LPPs do form complexes, but these do not seem to be specifically required for activity either in vitro or in vivo. Since neither dimerisation nor the C-terminus seem to be involved in substrate recognition, they may instead confer structural or functional stability through dimerisation. The results indicate that the associations we see are highly specific and occur only between monomers of the same protein.

Show MeSH
Related in: MedlinePlus