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Isoaspartate accumulation in mouse brain is associated with altered patterns of protein phosphorylation and acetylation, some of which are highly sex-dependent.

Qin Z, Kaufman RS, Khoury RN, Khoury MK, Aswad DW - PLoS ONE (2013)

Bottom Line: Average levels of dynamin-1 phosphorylation at Ser-778 and Ser-795 were higher in male KO vs.No changes in phosphorylation were found in synapsins I and II at Ser-603, in CRMP2 at Ser-522 or Thr-514, in DARPP-32 at Thr-34, or in PDK1 at Ser-241.We conclude that isoAsp accumulation is associated with altered functional status of several neuronal proteins that are highly susceptible to this type of damage.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology & Biochemistry, University of California Irvine, Irvine, California, United States of America.

ABSTRACT
Isoaspartate (isoAsp) formation is a major source of protein damage that is kept in check by the repair function of protein L-isoaspartyl methyltransferase (PIMT). Mice deficient in PIMT accumulate isoAsp-containing proteins, resulting in cognitive deficits, abnormal neuronal physiology and cytoarchitecture, and fatal epileptic seizures 30-60 days after birth. Synapsins I and II, dynamin-1, collapsin response mediator protein 2 (CRMP2), and α/β-tubulin are major targets of PIMT in brain. To investigate links between isoAsp accumulation and the neurological phenotype of the KO mice, we used Western blotting to compare patterns of in vivo phosphorylation or acetylation of the major PIMT targets listed above. Phosphorylations of synapsins I and II at Ser-9 were increased in female KO vs. WT mice, and acetylation of tubulin at Lys-40 was decreased in male KO vs. WT mice. Average levels of dynamin-1 phosphorylation at Ser-778 and Ser-795 were higher in male KO vs. WT mice, but the statistical significance (P>0.1) was low. No changes in phosphorylation were found in synapsins I and II at Ser-603, in CRMP2 at Ser-522 or Thr-514, in DARPP-32 at Thr-34, or in PDK1 at Ser-241. General levels of phosphorylation assessed with Pro-Q Diamond stain, or an anti-phosphotyrosine antibody, appeared similar in the WT and KO mice. We conclude that isoAsp accumulation is associated with altered functional status of several neuronal proteins that are highly susceptible to this type of damage. We also uncovered unexpected differences in how male and female mice respond to isoAsp accumulation in the brain.

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Phosphorylation of dynamin-1 at Ser-778 and Ser-795.Panels A-C show Western blots of brain extracts from female mice for dynamin-1 (A), phosphorylation of dynamin-1 at Ser-778 (B), and phosphorylation of dynamin-1 at Ser-795 (C). Panels D-F: Same as with A-C for male mice. Panel G: Quantitative measurements of band intensities after normalization to β-actin. Data are expressed as means ± SEM (n=5 for each genotype) with statistics as in Figure 1. None of the tests showed a significant difference.
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pone-0080758-g004: Phosphorylation of dynamin-1 at Ser-778 and Ser-795.Panels A-C show Western blots of brain extracts from female mice for dynamin-1 (A), phosphorylation of dynamin-1 at Ser-778 (B), and phosphorylation of dynamin-1 at Ser-795 (C). Panels D-F: Same as with A-C for male mice. Panel G: Quantitative measurements of band intensities after normalization to β-actin. Data are expressed as means ± SEM (n=5 for each genotype) with statistics as in Figure 1. None of the tests showed a significant difference.

Mentions: Dynamin-1 is a neuronal GTPase implicated in endocytosis of synaptic vesicles [35] and is another major in vivo substrate for PIMT [32]. Dynamin-1 function is regulated by phosphorylation via cyclin-dependent protein kinase 1 (Cdk1) at Ser-778, and protein kinase C (PKC) at Ser-795. Figure 4 shows the status of these phosphorylation sites in brain extracts of WT, HZ, and KO mice. The mean of ECL signals for KO males at both phosphorylation sites was increased by 75% over WT males; however the high variance of these signals resulted in a low level of statistical significance (P > 0.05). In contrast, the ECL signal at these phosphorylation sites for female WT and KO mice were nearly identical. No difference in dynamin-1 expression was seen in either sex. Firm conclusions from these results are hampered by the high mouse-to-mouse variability of dynamin-1 phosphorylation, but strongly suggests PIMT deficiency may increase dynamin-1 phosphorylation in males, and not in females. This is an interesting contrast to the results found with the synapsins.


Isoaspartate accumulation in mouse brain is associated with altered patterns of protein phosphorylation and acetylation, some of which are highly sex-dependent.

Qin Z, Kaufman RS, Khoury RN, Khoury MK, Aswad DW - PLoS ONE (2013)

Phosphorylation of dynamin-1 at Ser-778 and Ser-795.Panels A-C show Western blots of brain extracts from female mice for dynamin-1 (A), phosphorylation of dynamin-1 at Ser-778 (B), and phosphorylation of dynamin-1 at Ser-795 (C). Panels D-F: Same as with A-C for male mice. Panel G: Quantitative measurements of band intensities after normalization to β-actin. Data are expressed as means ± SEM (n=5 for each genotype) with statistics as in Figure 1. None of the tests showed a significant difference.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3818261&req=5

pone-0080758-g004: Phosphorylation of dynamin-1 at Ser-778 and Ser-795.Panels A-C show Western blots of brain extracts from female mice for dynamin-1 (A), phosphorylation of dynamin-1 at Ser-778 (B), and phosphorylation of dynamin-1 at Ser-795 (C). Panels D-F: Same as with A-C for male mice. Panel G: Quantitative measurements of band intensities after normalization to β-actin. Data are expressed as means ± SEM (n=5 for each genotype) with statistics as in Figure 1. None of the tests showed a significant difference.
Mentions: Dynamin-1 is a neuronal GTPase implicated in endocytosis of synaptic vesicles [35] and is another major in vivo substrate for PIMT [32]. Dynamin-1 function is regulated by phosphorylation via cyclin-dependent protein kinase 1 (Cdk1) at Ser-778, and protein kinase C (PKC) at Ser-795. Figure 4 shows the status of these phosphorylation sites in brain extracts of WT, HZ, and KO mice. The mean of ECL signals for KO males at both phosphorylation sites was increased by 75% over WT males; however the high variance of these signals resulted in a low level of statistical significance (P > 0.05). In contrast, the ECL signal at these phosphorylation sites for female WT and KO mice were nearly identical. No difference in dynamin-1 expression was seen in either sex. Firm conclusions from these results are hampered by the high mouse-to-mouse variability of dynamin-1 phosphorylation, but strongly suggests PIMT deficiency may increase dynamin-1 phosphorylation in males, and not in females. This is an interesting contrast to the results found with the synapsins.

Bottom Line: Average levels of dynamin-1 phosphorylation at Ser-778 and Ser-795 were higher in male KO vs.No changes in phosphorylation were found in synapsins I and II at Ser-603, in CRMP2 at Ser-522 or Thr-514, in DARPP-32 at Thr-34, or in PDK1 at Ser-241.We conclude that isoAsp accumulation is associated with altered functional status of several neuronal proteins that are highly susceptible to this type of damage.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology & Biochemistry, University of California Irvine, Irvine, California, United States of America.

ABSTRACT
Isoaspartate (isoAsp) formation is a major source of protein damage that is kept in check by the repair function of protein L-isoaspartyl methyltransferase (PIMT). Mice deficient in PIMT accumulate isoAsp-containing proteins, resulting in cognitive deficits, abnormal neuronal physiology and cytoarchitecture, and fatal epileptic seizures 30-60 days after birth. Synapsins I and II, dynamin-1, collapsin response mediator protein 2 (CRMP2), and α/β-tubulin are major targets of PIMT in brain. To investigate links between isoAsp accumulation and the neurological phenotype of the KO mice, we used Western blotting to compare patterns of in vivo phosphorylation or acetylation of the major PIMT targets listed above. Phosphorylations of synapsins I and II at Ser-9 were increased in female KO vs. WT mice, and acetylation of tubulin at Lys-40 was decreased in male KO vs. WT mice. Average levels of dynamin-1 phosphorylation at Ser-778 and Ser-795 were higher in male KO vs. WT mice, but the statistical significance (P>0.1) was low. No changes in phosphorylation were found in synapsins I and II at Ser-603, in CRMP2 at Ser-522 or Thr-514, in DARPP-32 at Thr-34, or in PDK1 at Ser-241. General levels of phosphorylation assessed with Pro-Q Diamond stain, or an anti-phosphotyrosine antibody, appeared similar in the WT and KO mice. We conclude that isoAsp accumulation is associated with altered functional status of several neuronal proteins that are highly susceptible to this type of damage. We also uncovered unexpected differences in how male and female mice respond to isoAsp accumulation in the brain.

Show MeSH
Related in: MedlinePlus