Limits...
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.

Show MeSH
Immunoprecipitation assays (a) Cell lysates co-transfected with WunM3 and WunGFP (first 3 lanes of each gel) or WunD2M3 and WunGFP (lanes 4, 5 and 6). 'Load' is unbound protein removed from the resin after the 2 hour incubation. 'Wash' is an aliquot of the final buffer change. 'Capture' is an aliquot of the resin. This shows capture of WunM3 and WunD2M3 by the resin in the left hand blot. In the right hand blot, however, WunGFP is only detected in the lane corresponding to the WunM3 capture: there is no WunGFP detectable in the lane corresponding to the WunD2M3 capture. This indicates an association between full length Wun that is abolished by the truncation. The lane from cells transfected with only WunGFP (lane 7) shows that the resin does not capture the GFP tag. (b) 1% triton X-100 breaks the association – WunM3 no longer pulls down WunGFP. (c) mLPP-1M3 is captured and pulls down mLPP-1GFP indicating an association. This is not seen between WunM3 and hLPP-3GFP, or mLPP-1M3 and WunGFP (d). TL refers to 'total lysate' – an aliquot of the complete lysate prior to incubation with the resin. (e) WunD:248>TM3 does not pull down WunGFP. (f) Incubation of WunM3 or WunGFP in PFA indicates monomers (M) at 39 KDa/51 KDa and dimers (D) at 70 KDa/100 KDa plus higher order oligomers. TL = total lysate not treated with PFA. (g) WunD2GFP and WunD:248>TGFP do not form oligomers with PFA, whereas hLPP-3GFP and mLPP-1M3 (h) do.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC319698&req=5

Figure 2: Immunoprecipitation assays (a) Cell lysates co-transfected with WunM3 and WunGFP (first 3 lanes of each gel) or WunD2M3 and WunGFP (lanes 4, 5 and 6). 'Load' is unbound protein removed from the resin after the 2 hour incubation. 'Wash' is an aliquot of the final buffer change. 'Capture' is an aliquot of the resin. This shows capture of WunM3 and WunD2M3 by the resin in the left hand blot. In the right hand blot, however, WunGFP is only detected in the lane corresponding to the WunM3 capture: there is no WunGFP detectable in the lane corresponding to the WunD2M3 capture. This indicates an association between full length Wun that is abolished by the truncation. The lane from cells transfected with only WunGFP (lane 7) shows that the resin does not capture the GFP tag. (b) 1% triton X-100 breaks the association – WunM3 no longer pulls down WunGFP. (c) mLPP-1M3 is captured and pulls down mLPP-1GFP indicating an association. This is not seen between WunM3 and hLPP-3GFP, or mLPP-1M3 and WunGFP (d). TL refers to 'total lysate' – an aliquot of the complete lysate prior to incubation with the resin. (e) WunD:248>TM3 does not pull down WunGFP. (f) Incubation of WunM3 or WunGFP in PFA indicates monomers (M) at 39 KDa/51 KDa and dimers (D) at 70 KDa/100 KDa plus higher order oligomers. TL = total lysate not treated with PFA. (g) WunD2GFP and WunD:248>TGFP do not form oligomers with PFA, whereas hLPP-3GFP and mLPP-1M3 (h) do.

Mentions: We investigated interactions between Wun monomers using an anti-myc immunoaffinity resin. S2 cells co-transfected with WunM3, WunGFP plus Actin5C-GAL4 were lysed in 0.1% triton X-100 buffer and incubated with the resin before extensive washing to remove unbound protein. The resin was then subjected to Western analysis (Fig. 2a). No WunGFP is detected in the final wash but a strong signal is seen in the lane containing the resin. As the resin selectively captures myc epitope tagged proteins, the WunGFP must be associating with the captured WunM3. We conclude that wild-type Wun can interact with itself and form complexes. Resin incubated with WunGFP alone shows no capture confirming that the resin does not recognise the GFP epitope. The protocol was repeated with 1% triton X-100. The results show loss of association – no WunGFP is detectable, whilst WunM3 is still captured (Fig. 2b). This again confirms that the resin does not recognise GFP tagged proteins, and indicates that the association between Wun monomers can be disrupted by increasing the concentration of detergent in the extraction buffer. We examined the specificity of the interaction in 0.1% triton lysis buffer, and found that Wun does not form associations with its closely related counterpart Wun-2 (data not shown). Furthermore, whilst mLPP-1 and hLPP-3 can both self-interact (Fig. 2c), neither of them associate with Wun (Fig. 2d). This indicates that what we observe is a highly specific interaction which can not occur between closely related but non-identical proteins. We tried several times to investigate interactions between monomers of Wunen-2, but were unable to either express the protein at adequate levels, or to capture that protein that was expressed.


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)

Immunoprecipitation assays (a) Cell lysates co-transfected with WunM3 and WunGFP (first 3 lanes of each gel) or WunD2M3 and WunGFP (lanes 4, 5 and 6). 'Load' is unbound protein removed from the resin after the 2 hour incubation. 'Wash' is an aliquot of the final buffer change. 'Capture' is an aliquot of the resin. This shows capture of WunM3 and WunD2M3 by the resin in the left hand blot. In the right hand blot, however, WunGFP is only detected in the lane corresponding to the WunM3 capture: there is no WunGFP detectable in the lane corresponding to the WunD2M3 capture. This indicates an association between full length Wun that is abolished by the truncation. The lane from cells transfected with only WunGFP (lane 7) shows that the resin does not capture the GFP tag. (b) 1% triton X-100 breaks the association – WunM3 no longer pulls down WunGFP. (c) mLPP-1M3 is captured and pulls down mLPP-1GFP indicating an association. This is not seen between WunM3 and hLPP-3GFP, or mLPP-1M3 and WunGFP (d). TL refers to 'total lysate' – an aliquot of the complete lysate prior to incubation with the resin. (e) WunD:248>TM3 does not pull down WunGFP. (f) Incubation of WunM3 or WunGFP in PFA indicates monomers (M) at 39 KDa/51 KDa and dimers (D) at 70 KDa/100 KDa plus higher order oligomers. TL = total lysate not treated with PFA. (g) WunD2GFP and WunD:248>TGFP do not form oligomers with PFA, whereas hLPP-3GFP and mLPP-1M3 (h) do.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Immunoprecipitation assays (a) Cell lysates co-transfected with WunM3 and WunGFP (first 3 lanes of each gel) or WunD2M3 and WunGFP (lanes 4, 5 and 6). 'Load' is unbound protein removed from the resin after the 2 hour incubation. 'Wash' is an aliquot of the final buffer change. 'Capture' is an aliquot of the resin. This shows capture of WunM3 and WunD2M3 by the resin in the left hand blot. In the right hand blot, however, WunGFP is only detected in the lane corresponding to the WunM3 capture: there is no WunGFP detectable in the lane corresponding to the WunD2M3 capture. This indicates an association between full length Wun that is abolished by the truncation. The lane from cells transfected with only WunGFP (lane 7) shows that the resin does not capture the GFP tag. (b) 1% triton X-100 breaks the association – WunM3 no longer pulls down WunGFP. (c) mLPP-1M3 is captured and pulls down mLPP-1GFP indicating an association. This is not seen between WunM3 and hLPP-3GFP, or mLPP-1M3 and WunGFP (d). TL refers to 'total lysate' – an aliquot of the complete lysate prior to incubation with the resin. (e) WunD:248>TM3 does not pull down WunGFP. (f) Incubation of WunM3 or WunGFP in PFA indicates monomers (M) at 39 KDa/51 KDa and dimers (D) at 70 KDa/100 KDa plus higher order oligomers. TL = total lysate not treated with PFA. (g) WunD2GFP and WunD:248>TGFP do not form oligomers with PFA, whereas hLPP-3GFP and mLPP-1M3 (h) do.
Mentions: We investigated interactions between Wun monomers using an anti-myc immunoaffinity resin. S2 cells co-transfected with WunM3, WunGFP plus Actin5C-GAL4 were lysed in 0.1% triton X-100 buffer and incubated with the resin before extensive washing to remove unbound protein. The resin was then subjected to Western analysis (Fig. 2a). No WunGFP is detected in the final wash but a strong signal is seen in the lane containing the resin. As the resin selectively captures myc epitope tagged proteins, the WunGFP must be associating with the captured WunM3. We conclude that wild-type Wun can interact with itself and form complexes. Resin incubated with WunGFP alone shows no capture confirming that the resin does not recognise the GFP epitope. The protocol was repeated with 1% triton X-100. The results show loss of association – no WunGFP is detectable, whilst WunM3 is still captured (Fig. 2b). This again confirms that the resin does not recognise GFP tagged proteins, and indicates that the association between Wun monomers can be disrupted by increasing the concentration of detergent in the extraction buffer. We examined the specificity of the interaction in 0.1% triton lysis buffer, and found that Wun does not form associations with its closely related counterpart Wun-2 (data not shown). Furthermore, whilst mLPP-1 and hLPP-3 can both self-interact (Fig. 2c), neither of them associate with Wun (Fig. 2d). This indicates that what we observe is a highly specific interaction which can not occur between closely related but non-identical proteins. We tried several times to investigate interactions between monomers of Wunen-2, but were unable to either express the protein at adequate levels, or to capture that protein that was expressed.

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