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The multifunctional poly(A)-binding protein (PABP) 1 is subject to extensive dynamic post-translational modification, which molecular modelling suggests plays an important role in co-ordinating its activities.

Brook M, McCracken L, Reddington JP, Lu ZL, Morrice NA, Gray NK - Biochem. J. (2012)

Bottom Line: Intriguingly, PABP1 contains glutamate and aspartate methylations, modifications of unknown function in eukaryotes, as well as lysine and arginine methylations, and lysine acetylations.The latter dramatically alter the pI of PABP1, an effect also observed during the cell cycle, suggesting that different biological processes/stimuli can regulate its modification status, although PABP1 also probably exists in differentially modified subpopulations within cells.Modelling using available structures implicates these modifications in regulating interactions with individual PAM2 (PABP-interacting motif 2)-containing proteins, suggesting a direct link between PABP1 modification status and the formation of distinct mRNP (messenger ribonucleoprotein) complexes that regulate mRNA fate in the cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Reproductive Health/MRC Human Reproductive Sciences Unit, Queen's Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, Scotland, UK. matt.brook@ed.ac.uk

ABSTRACT
PABP1 [poly(A)-binding protein 1] is a central regulator of mRNA translation and stability and is required for miRNA (microRNA)-mediated regulation and nonsense-mediated decay. Numerous protein, as well as RNA, interactions underlie its multi-functional nature; however, it is unclear how its different activities are co-ordinated, since many partners interact via overlapping binding sites. In the present study, we show that human PABP1 is subject to elaborate post-translational modification, identifying 14 modifications located throughout the functional domains, all but one of which are conserved in mouse. Intriguingly, PABP1 contains glutamate and aspartate methylations, modifications of unknown function in eukaryotes, as well as lysine and arginine methylations, and lysine acetylations. The latter dramatically alter the pI of PABP1, an effect also observed during the cell cycle, suggesting that different biological processes/stimuli can regulate its modification status, although PABP1 also probably exists in differentially modified subpopulations within cells. Two lysine residues were differentially acetylated or methylated, revealing that PABP1 may be the first example of a cytoplasmic protein utilizing a 'methylation/acetylation switch'. Modelling using available structures implicates these modifications in regulating interactions with individual PAM2 (PABP-interacting motif 2)-containing proteins, suggesting a direct link between PABP1 modification status and the formation of distinct mRNP (messenger ribonucleoprotein) complexes that regulate mRNA fate in the cytoplasm.

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The pI of PABP1 is dynamically regulated during the cell cycle and is modified by lysine acetylation(A) HeLa cells were either left untreated or treated with 400 nM TSA/5 mM nicotinamide. Cell extracts were fractionated using a pH 3–10 linear immobilized pH gradient (IPG) and immunoblotted for PABP1 and GAPDH. (B) HeLa cells were untreated (Asynch) or synchronized in S- or G2/M-phase and cell extracts were fractionated and immunoblotted as described in (A). (A and B) GAPDH [41] exhibits the expected pI distribution for its unmodified and modified forms.
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Figure 7: The pI of PABP1 is dynamically regulated during the cell cycle and is modified by lysine acetylation(A) HeLa cells were either left untreated or treated with 400 nM TSA/5 mM nicotinamide. Cell extracts were fractionated using a pH 3–10 linear immobilized pH gradient (IPG) and immunoblotted for PABP1 and GAPDH. (B) HeLa cells were untreated (Asynch) or synchronized in S- or G2/M-phase and cell extracts were fractionated and immunoblotted as described in (A). (A and B) GAPDH [41] exhibits the expected pI distribution for its unmodified and modified forms.

Mentions: Interestingly, treatment of HeLa cells with TSA and nicotinamide, which blocks lysine deacetylation, resulted in a significant change in the proportion of PABP1 in the pI range ~pH 5.9–8.3 (Figure 7A). This suggests that, although apparently sub-stoichiometric in asynchronously growing HeLa cells, lysine acetylation could act as a major contributor to the overall charge state of human PABP1. Thus to assess whether such changes in PABP1 pI occur under physiological conditions, we examined its modification status during cell division, when extensive translational regulation is manifest [28]. In asynchronous HeLa cells the pI of the majority of PABP1 is in the pH 8.8–10 range, as expected (Figure 7B, lanes 11 and 12). However, in double-thymidine-synchronized S-phase HeLa cells PABP1 is detected with a pI range ~pH 5.9–10. The observed reduction in PABP1 pI is further enhanced in double-thymidine/nocodazole-synchronized G2/M-phase HeLa cells, where PABP1 is no longer detected in the pI range ~pH 8.8–10 with the bulk of PABP1 appearing within the pI range ~pH 5.3–8.25, consistent with a high level of de novo post-translational modification. Taken together these data reveal that PABP1 is subject to complex and dynamic post-translational modification during the mitotic cell cycle, of which lysine acetylation may be an important component.


The multifunctional poly(A)-binding protein (PABP) 1 is subject to extensive dynamic post-translational modification, which molecular modelling suggests plays an important role in co-ordinating its activities.

Brook M, McCracken L, Reddington JP, Lu ZL, Morrice NA, Gray NK - Biochem. J. (2012)

The pI of PABP1 is dynamically regulated during the cell cycle and is modified by lysine acetylation(A) HeLa cells were either left untreated or treated with 400 nM TSA/5 mM nicotinamide. Cell extracts were fractionated using a pH 3–10 linear immobilized pH gradient (IPG) and immunoblotted for PABP1 and GAPDH. (B) HeLa cells were untreated (Asynch) or synchronized in S- or G2/M-phase and cell extracts were fractionated and immunoblotted as described in (A). (A and B) GAPDH [41] exhibits the expected pI distribution for its unmodified and modified forms.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3298439&req=5

Figure 7: The pI of PABP1 is dynamically regulated during the cell cycle and is modified by lysine acetylation(A) HeLa cells were either left untreated or treated with 400 nM TSA/5 mM nicotinamide. Cell extracts were fractionated using a pH 3–10 linear immobilized pH gradient (IPG) and immunoblotted for PABP1 and GAPDH. (B) HeLa cells were untreated (Asynch) or synchronized in S- or G2/M-phase and cell extracts were fractionated and immunoblotted as described in (A). (A and B) GAPDH [41] exhibits the expected pI distribution for its unmodified and modified forms.
Mentions: Interestingly, treatment of HeLa cells with TSA and nicotinamide, which blocks lysine deacetylation, resulted in a significant change in the proportion of PABP1 in the pI range ~pH 5.9–8.3 (Figure 7A). This suggests that, although apparently sub-stoichiometric in asynchronously growing HeLa cells, lysine acetylation could act as a major contributor to the overall charge state of human PABP1. Thus to assess whether such changes in PABP1 pI occur under physiological conditions, we examined its modification status during cell division, when extensive translational regulation is manifest [28]. In asynchronous HeLa cells the pI of the majority of PABP1 is in the pH 8.8–10 range, as expected (Figure 7B, lanes 11 and 12). However, in double-thymidine-synchronized S-phase HeLa cells PABP1 is detected with a pI range ~pH 5.9–10. The observed reduction in PABP1 pI is further enhanced in double-thymidine/nocodazole-synchronized G2/M-phase HeLa cells, where PABP1 is no longer detected in the pI range ~pH 8.8–10 with the bulk of PABP1 appearing within the pI range ~pH 5.3–8.25, consistent with a high level of de novo post-translational modification. Taken together these data reveal that PABP1 is subject to complex and dynamic post-translational modification during the mitotic cell cycle, of which lysine acetylation may be an important component.

Bottom Line: Intriguingly, PABP1 contains glutamate and aspartate methylations, modifications of unknown function in eukaryotes, as well as lysine and arginine methylations, and lysine acetylations.The latter dramatically alter the pI of PABP1, an effect also observed during the cell cycle, suggesting that different biological processes/stimuli can regulate its modification status, although PABP1 also probably exists in differentially modified subpopulations within cells.Modelling using available structures implicates these modifications in regulating interactions with individual PAM2 (PABP-interacting motif 2)-containing proteins, suggesting a direct link between PABP1 modification status and the formation of distinct mRNP (messenger ribonucleoprotein) complexes that regulate mRNA fate in the cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Reproductive Health/MRC Human Reproductive Sciences Unit, Queen's Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, Scotland, UK. matt.brook@ed.ac.uk

ABSTRACT
PABP1 [poly(A)-binding protein 1] is a central regulator of mRNA translation and stability and is required for miRNA (microRNA)-mediated regulation and nonsense-mediated decay. Numerous protein, as well as RNA, interactions underlie its multi-functional nature; however, it is unclear how its different activities are co-ordinated, since many partners interact via overlapping binding sites. In the present study, we show that human PABP1 is subject to elaborate post-translational modification, identifying 14 modifications located throughout the functional domains, all but one of which are conserved in mouse. Intriguingly, PABP1 contains glutamate and aspartate methylations, modifications of unknown function in eukaryotes, as well as lysine and arginine methylations, and lysine acetylations. The latter dramatically alter the pI of PABP1, an effect also observed during the cell cycle, suggesting that different biological processes/stimuli can regulate its modification status, although PABP1 also probably exists in differentially modified subpopulations within cells. Two lysine residues were differentially acetylated or methylated, revealing that PABP1 may be the first example of a cytoplasmic protein utilizing a 'methylation/acetylation switch'. Modelling using available structures implicates these modifications in regulating interactions with individual PAM2 (PABP-interacting motif 2)-containing proteins, suggesting a direct link between PABP1 modification status and the formation of distinct mRNP (messenger ribonucleoprotein) complexes that regulate mRNA fate in the cytoplasm.

Show MeSH
Related in: MedlinePlus