Limits...
Differences in specificity and selectivity between CBP and p300 acetylation of histone H3 and H3/H4.

Henry RA, Kuo YM, Andrews AJ - Biochemistry (2013)

Bottom Line: With limiting tetramer, CBP displays higher specificities, especially at H3K18, where CBP specificity is 10³²-fold higher than p300.With limiting acetyl-CoA, p300 has the highest specificity at H4K16, where specificity is 10¹⁸-fold higher than CBP.This discovery of unique specificity for targets of CBP- vs p300-mediated acetylation of histone lysine residues presents a new model for understanding their respective biological roles and possibly an opportunity for selective therapeutic intervention.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States.

ABSTRACT
Although p300 and CBP lysine acetyltransferases are often treated interchangeably, the inability of one enzyme to compensate for the loss of the other suggests unique roles for each. As these deficiencies coincide with aberrant levels of histone acetylation, we hypothesized that the key difference between p300 and CBP activity is differences in their specificity/selectivity for lysines within the histones. Utilizing a label-free, quantitative mass spectrometry based technique, we determined the kinetic parameters of both CBP and p300 at each lysine of H3 and H4, under conditions we would expect to encounter in the cell (either limiting acetyl-CoA or histone). Our results show that while p300 and CBP acetylate many common residues on H3 and H4, they do in fact possess very different specificities, and these specificities are dependent on whether histone or acetyl-CoA is limiting. Steady-state experiments with limiting H3 demonstrate that both CBP and p300 acetylate H3K14, H3K18, H3K23, with p300 having specificities up to 10¹⁰-fold higher than CBP. Utilizing tetramer as a substrate, both enzymes also acetylate H4K5, H4K8, H4K12, and H4K16. With limiting tetramer, CBP displays higher specificities, especially at H3K18, where CBP specificity is 10³²-fold higher than p300. With limiting acetyl-CoA, p300 has the highest specificity at H4K16, where specificity is 10¹⁸-fold higher than CBP. This discovery of unique specificity for targets of CBP- vs p300-mediated acetylation of histone lysine residues presents a new model for understanding their respective biological roles and possibly an opportunity for selective therapeutic intervention.

Show MeSH

Related in: MedlinePlus

Determinationof steady-state kinetic parameters of CBP- and p300-mediatedacetylation of histone H3 when titrating acetyl-CoA. Experiments wereperformed at 37 °C in 100 mM ammonium bicarbonate and 50 mM HEPESbuffer (pH 7.8) at 37 °C. Assays for p300 contained 50 nM p300,17.5 μM H3, and varying concentrations of acetyl-CoA (1–200μM). Assays for CBP contained 7 nM CBP, 7.5 μM H3, andvarying concentrations of acetyl-CoA (1–200 μM). Experimentswere quenched with 4 vol of TCA and boiled at 95 °C for 5 min.Sites displaying the highest specificity (kcat/KmnH) foreither CBP or p300 where chosen for representative graphs. (A) Nonlinearfit of CBP acetylation of histone H3K14. (B) Nonlinear fit of p300acetylation of histone H3K14. (C) Comparison of the specificity constants(kcat/KmnH) of CBP (black) and p300 (gray) on H3K9,H3K14, H3K18, and H3K23. (D) The log of the ratio of specificity (CBP/p300)between CBP and p300 at each site of H3. All quantified sites canbe found in Supplemental Figure 2. Theapparent kinetic parameters are summarized in Table 3.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3756530&req=5

fig2: Determinationof steady-state kinetic parameters of CBP- and p300-mediatedacetylation of histone H3 when titrating acetyl-CoA. Experiments wereperformed at 37 °C in 100 mM ammonium bicarbonate and 50 mM HEPESbuffer (pH 7.8) at 37 °C. Assays for p300 contained 50 nM p300,17.5 μM H3, and varying concentrations of acetyl-CoA (1–200μM). Assays for CBP contained 7 nM CBP, 7.5 μM H3, andvarying concentrations of acetyl-CoA (1–200 μM). Experimentswere quenched with 4 vol of TCA and boiled at 95 °C for 5 min.Sites displaying the highest specificity (kcat/KmnH) foreither CBP or p300 where chosen for representative graphs. (A) Nonlinearfit of CBP acetylation of histone H3K14. (B) Nonlinear fit of p300acetylation of histone H3K14. (C) Comparison of the specificity constants(kcat/KmnH) of CBP (black) and p300 (gray) on H3K9,H3K14, H3K18, and H3K23. (D) The log of the ratio of specificity (CBP/p300)between CBP and p300 at each site of H3. All quantified sites canbe found in Supplemental Figure 2. Theapparent kinetic parameters are summarized in Table 3.

Mentions: Next we measured the steady-stateparameters for acetyl-CoA undersaturating histone H3 concentrations (10–15 μM) (Figures 2A,B and S2). These experimentsare more complicated in the fact that, for concentrations of acetyl-CoAthat are less than the concentration of H3, we have to measure totalacetylation less than 0.1 times the total concentration of acetyl-CoAtimes the concentration of histone (see ref (27) for details). These experimentsallowed us to determine if limiting the amount of acetyl-CoA availableto either CBP or p300 would affect their specificity. The amount ofacetyl-CoA used for these experiments falls well within previouslyreported ranges for cellular acetyl-CoA concentrations.34 Interestingly, the order of specificity changedfrom what we observed in titrating H3. For CBP we found that the orderof kcat/K1/2 is K18 > K14 > K23 with a range of 7–40-fold differenceinspecificity. The Hill coefficient changes this order again, wherethe order of specificity based on kcat/K1/2nH isK14 > K23 > K18 (Figure 2C); the movementofK18 from the first position to last is due to the large Hill coefficientfor K14 (∼6). While we did not observe a change in the orderof the kcat/K1/2 for p300 (K18 > K14 > K23 > K9), when we compare the kcat/K1/2nH to that of the H3 titration, we observe a changein the orderfor the last two positions to (K14 > K18 > K23 > K9) (Figure 2C). This results in a difference in specificityof up to 1042-fold. This is in contrast with what we previouslyobserved for Gcn5, where the order of acetylation was unchanged foreither limiting acetyl-CoA or H3.27 Catalyticproficiency or ((kcat/K1/2nH)/knE) for p300 goes as high as 1045 and 1032 for CBP. The differences in specificity between CBP and p300 arealso more exaggerated: with acetyl-CoA p300 has an advantage of ∼1015-fold for K14 and K18, and CBP has a ∼105-fold advantage for K23 (Figure 2D). Thisvalue is too large to know for K9. The kinetic parameters for theseexperiments are summarized in Table 3.


Differences in specificity and selectivity between CBP and p300 acetylation of histone H3 and H3/H4.

Henry RA, Kuo YM, Andrews AJ - Biochemistry (2013)

Determinationof steady-state kinetic parameters of CBP- and p300-mediatedacetylation of histone H3 when titrating acetyl-CoA. Experiments wereperformed at 37 °C in 100 mM ammonium bicarbonate and 50 mM HEPESbuffer (pH 7.8) at 37 °C. Assays for p300 contained 50 nM p300,17.5 μM H3, and varying concentrations of acetyl-CoA (1–200μM). Assays for CBP contained 7 nM CBP, 7.5 μM H3, andvarying concentrations of acetyl-CoA (1–200 μM). Experimentswere quenched with 4 vol of TCA and boiled at 95 °C for 5 min.Sites displaying the highest specificity (kcat/KmnH) foreither CBP or p300 where chosen for representative graphs. (A) Nonlinearfit of CBP acetylation of histone H3K14. (B) Nonlinear fit of p300acetylation of histone H3K14. (C) Comparison of the specificity constants(kcat/KmnH) of CBP (black) and p300 (gray) on H3K9,H3K14, H3K18, and H3K23. (D) The log of the ratio of specificity (CBP/p300)between CBP and p300 at each site of H3. All quantified sites canbe found in Supplemental Figure 2. Theapparent kinetic parameters are summarized in Table 3.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3756530&req=5

fig2: Determinationof steady-state kinetic parameters of CBP- and p300-mediatedacetylation of histone H3 when titrating acetyl-CoA. Experiments wereperformed at 37 °C in 100 mM ammonium bicarbonate and 50 mM HEPESbuffer (pH 7.8) at 37 °C. Assays for p300 contained 50 nM p300,17.5 μM H3, and varying concentrations of acetyl-CoA (1–200μM). Assays for CBP contained 7 nM CBP, 7.5 μM H3, andvarying concentrations of acetyl-CoA (1–200 μM). Experimentswere quenched with 4 vol of TCA and boiled at 95 °C for 5 min.Sites displaying the highest specificity (kcat/KmnH) foreither CBP or p300 where chosen for representative graphs. (A) Nonlinearfit of CBP acetylation of histone H3K14. (B) Nonlinear fit of p300acetylation of histone H3K14. (C) Comparison of the specificity constants(kcat/KmnH) of CBP (black) and p300 (gray) on H3K9,H3K14, H3K18, and H3K23. (D) The log of the ratio of specificity (CBP/p300)between CBP and p300 at each site of H3. All quantified sites canbe found in Supplemental Figure 2. Theapparent kinetic parameters are summarized in Table 3.
Mentions: Next we measured the steady-stateparameters for acetyl-CoA undersaturating histone H3 concentrations (10–15 μM) (Figures 2A,B and S2). These experimentsare more complicated in the fact that, for concentrations of acetyl-CoAthat are less than the concentration of H3, we have to measure totalacetylation less than 0.1 times the total concentration of acetyl-CoAtimes the concentration of histone (see ref (27) for details). These experimentsallowed us to determine if limiting the amount of acetyl-CoA availableto either CBP or p300 would affect their specificity. The amount ofacetyl-CoA used for these experiments falls well within previouslyreported ranges for cellular acetyl-CoA concentrations.34 Interestingly, the order of specificity changedfrom what we observed in titrating H3. For CBP we found that the orderof kcat/K1/2 is K18 > K14 > K23 with a range of 7–40-fold differenceinspecificity. The Hill coefficient changes this order again, wherethe order of specificity based on kcat/K1/2nH isK14 > K23 > K18 (Figure 2C); the movementofK18 from the first position to last is due to the large Hill coefficientfor K14 (∼6). While we did not observe a change in the orderof the kcat/K1/2 for p300 (K18 > K14 > K23 > K9), when we compare the kcat/K1/2nH to that of the H3 titration, we observe a changein the orderfor the last two positions to (K14 > K18 > K23 > K9) (Figure 2C). This results in a difference in specificityof up to 1042-fold. This is in contrast with what we previouslyobserved for Gcn5, where the order of acetylation was unchanged foreither limiting acetyl-CoA or H3.27 Catalyticproficiency or ((kcat/K1/2nH)/knE) for p300 goes as high as 1045 and 1032 for CBP. The differences in specificity between CBP and p300 arealso more exaggerated: with acetyl-CoA p300 has an advantage of ∼1015-fold for K14 and K18, and CBP has a ∼105-fold advantage for K23 (Figure 2D). Thisvalue is too large to know for K9. The kinetic parameters for theseexperiments are summarized in Table 3.

Bottom Line: With limiting tetramer, CBP displays higher specificities, especially at H3K18, where CBP specificity is 10³²-fold higher than p300.With limiting acetyl-CoA, p300 has the highest specificity at H4K16, where specificity is 10¹⁸-fold higher than CBP.This discovery of unique specificity for targets of CBP- vs p300-mediated acetylation of histone lysine residues presents a new model for understanding their respective biological roles and possibly an opportunity for selective therapeutic intervention.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States.

ABSTRACT
Although p300 and CBP lysine acetyltransferases are often treated interchangeably, the inability of one enzyme to compensate for the loss of the other suggests unique roles for each. As these deficiencies coincide with aberrant levels of histone acetylation, we hypothesized that the key difference between p300 and CBP activity is differences in their specificity/selectivity for lysines within the histones. Utilizing a label-free, quantitative mass spectrometry based technique, we determined the kinetic parameters of both CBP and p300 at each lysine of H3 and H4, under conditions we would expect to encounter in the cell (either limiting acetyl-CoA or histone). Our results show that while p300 and CBP acetylate many common residues on H3 and H4, they do in fact possess very different specificities, and these specificities are dependent on whether histone or acetyl-CoA is limiting. Steady-state experiments with limiting H3 demonstrate that both CBP and p300 acetylate H3K14, H3K18, H3K23, with p300 having specificities up to 10¹⁰-fold higher than CBP. Utilizing tetramer as a substrate, both enzymes also acetylate H4K5, H4K8, H4K12, and H4K16. With limiting tetramer, CBP displays higher specificities, especially at H3K18, where CBP specificity is 10³²-fold higher than p300. With limiting acetyl-CoA, p300 has the highest specificity at H4K16, where specificity is 10¹⁸-fold higher than CBP. This discovery of unique specificity for targets of CBP- vs p300-mediated acetylation of histone lysine residues presents a new model for understanding their respective biological roles and possibly an opportunity for selective therapeutic intervention.

Show MeSH
Related in: MedlinePlus