A quantitative study on the in vitro and in vivo acetylation of high mobility group A1 proteins.
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.
Zhang
Qingchun
Q
Department of Chemistry-027, University of California, Riverside, California 92521-0403, USA.
Zhang
Kangling
K
Zou
Yan
Y
Perna
Avi
A
Wang
Yinsheng
Y
D000107:Acetylation; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D025741:HMGA1a Protein / Q000737:chemistry / Q000378:metabolism*; D025742:HMGA1b Protein / Q000737:chemistry / Q000378:metabolism*; D051548:Histone Acetyltransferases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008297:Male; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000737:chemistry* / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D009363:Neoplasm Proteins; D011506:Proteins; D025742:HMGA1b Protein; D025741:HMGA1a Protein; D051548:Histone Acetyltransferases