A quantitative study on the in vitro and in vivo acetylation of high mobility group A1 proteins

Abstract

High mobility group (HMG) A1 proteins are subject to a number of post-translational modifications, which may regulate their function in gene transcription and other cellular processes. We examined, by using mass spectrometry, the acetylation of HMGA1a and HMGA1b proteins induced by histone acetyltransferases p300 and PCAF in vitro and in PC-3 human prostate cancer cells in vivo. It turned out that five lysine residues in HMGA1a, i.e., Lys-14, Lys-64, Lys-66, Lys-70, and Lys-73, could be acetylated by both p300 and PCAF. We further quantified the level of acetylation by analyzing, with LC-MS/MS, the proteolytic peptides of the in vitro or in vivo acetylated HMGA1 proteins where the unmodified lysine residues were chemically derivatized with a perdeuterated acetyl group. Quantification results revealed that p300 and PCAF exhibited different site preferences for the acetylation; the preference of p300 acetylation followed the order of Lys-64 approximately Lys-70 > Lys-66 > Lys-14 approximately Lys73, whereas the selectivity of PCAF acetylation followed the sequence of Lys-70 approximately Lys-73 > Lys-64 approximately Lys-66 > Lys-14. HMGA1b was acetylated in a very similar fashion as HMGA1a. We also demonstrated that C-terminal phosphorylation of HMGA1 proteins did not affect the in vitro acetylation of the two proteins by either p300 or PCAF. Moreover, we examined the acetylation of lysine residues in HMGA1a and HMGA1b isolated from PC-3 human prostate cancer cells. Our results showed that all the above five lysine residues were also acetylated in vivo, with Lys-64, Lys-66 and Lys-70 in HMGA1a exhibiting higher levels of acetylation than Lys-14 and Lys-73.

Figure 1

The sequences of HMGA1a and HMGA1b. The acetylated lysine residues identified in this study were highlighted in bold, and peptides containing these lysine residues were underlined.

Figure 2

An outline of the experimental approach employed to quantify the in-vitro and in-vivo acetylation of HMGA1a and HMGA1b. HMGA1 proteins were treated with deuterated acetic anhydride and deuterated acetic acid for 14 h. As a result, each of the unacetylated lysine residues was conjugated with a deuterated acetyl group. Digesting the proteins with trypsin or Glu-C produced the desired peptides, in which the acetylated lysine residue (either endogenously acetylated in PC-3 cells or in-vitro acetylated by p300 or PCAF) was tagged with a protiated acetyl moiety (H₃-Ac) and the unacetylated lysine residue was attached with a deuterated acetyl group (D₃-Ac). Since the mass difference between H₃-Ac and D₃-Ac is 3 Da, the acetylation level can be determined from the ratio of the abundance of ions bearing H₃-Ac over the sum of those bearing H₃-Ac and D₃-Ac.

Figure 3

ESI mass spectra of tryptic peptide K₇₃TTTTPGR from HMGA1a acetylated by histone acetyltransferases PCAF (A) and p300 (B).

Figure 4

Product-ion spectra of the ESI-produced [M + 2H]²⁺ ions of tryptic peptide K₇₃TTTTPGR of HMGA1a that was acetylated by PCAF (A) or p300 (B) in vitro. The two insets on the right, showing two groups of peaks, are the expanded mass spectra of the parent ion m/z 452.3 (453.8), corresponding to the unacetylated and monoacetylated forms of this peptide induced by PCAF (top) and p300 (bottom), respectively. The symbol “Ac” designates ions bearing acetyl lysine residues induced by histone acetyltransferases. The two insets on the left are the expanded mass spectra of the b₂ ion, corresponding to the acetylated (H₃-Ac) and unacetylated (D₃-Ac) forms of this product ion, in which the acetylation was induced by PCAF (top) or p300 (bottom). A scheme summarizing the observed fragment ions of this peptide is shown above the mass spectra.

Figure 5

ESI mass spectra of tryptic peptide GRPKGSK₆₄NK₆₆GAAK₇₀TR from HMGA1a acetylated by histone acetyltransferases PCAF (A) and p300 (B).

Figure 6

Product-ion spectra of the ESI-produced [M + 2H]²⁺ ion of the tryptic peptide GRPKGSK₆₄NK₆₆GAAK₇₀TR from HMGA1a that was acetylated in vitro by PCAF (A) and p300 (B). The four insets are the expanded mass spectra of y₆ (left) and y₇ (right) ions, in which the acetylation was induced by PCAF (top) and p300 (bottom). A scheme summarizing the observed fragment ions of this peptide is shown above the mass spectra.

Figure 7

Histograms showing the fraction of in-vitro acetylation at Lys-14, Lys-64, Lys-66, Lys-70 and Lys-73 of recombinant HMGA1a and phosphorylated HMGA1a (phos-HMGA1a) catalyzed by both p300 and PCAF (top panel), and the fraction of in-vitro acetylation at Lys-14, Lys-53, Lys-55 Lys-59 and Lys-62 of recombinant HMGA1b and phosphorylated HMGA1b (phos-HMGA1b) catalyzed by both p300 and PCAF (bottom panel).

Figure 8

Fractions of acetylation at residues Lys-14, Lys-64, Lys-66, Lys-70, Lys-73 of HMGA1a and at residues Lys-14, Lys-53, Lys-55, Lys-59, Lys-62 of HMGA1b purified from PC-3 human prostate cancer cells.