Constitutive and dynamic phosphorylation and acetylation sites on NUCKS, a hypermodified nuclear protein, studied by quantitative proteomics.
Nuclear ubiquitous casein and cyclin-dependent kinases substrate (NUCKS) is a 27 kDa chromosomal protein of unknown function. Its amino acid composition as well as the structure of its DNA binding domain resembles that of high mobility group A (HMGA) proteins, chromosomal proteins known as modulators of chromatin conformation and regulators of transcription. Conformation and function of the HMGA proteins are regulated by phosphorylation and acetylation. So far 19 phosphorylation sites had been reported in NUCKS. In this study, we have identified all known and six additional phosphorylation sites, and also mapped multiple sites of acetylation, methylation and formylation. We measured cell cycle dependent changes of phosphorylation and acetylation of NUCKS in HeLa cells through stable isotope labeling by amino acids in cell culture (SILAC), using the dephosphorylated protein for normalization. We identified sites that were highly phosphorylated or dephosphorylated in mitotically arrested cells as well as sites that were constitutively phosphorylated. The extent of acetylation is reduced in mitotically and G1 arrested cells. Analysis of human cancer specimens revealed that in tissues the extent of acetylation, formylation and methylation is higher than in cultured cells. In breast cancer samples, seven acetylation, three methylation, and three formylation sites were mapped in NUCKS. Of the 243 amino acids, at least 36 can be modified with a total of 57 posttranslational modifications. Thus, NUCKS appears to have the highest ratio of modified to unmodified residues of any protein so far described.
Wiśniewski
Jacek R
JR
Proteomics and Signal Transduction, Max-Planck Institute for Biochemistry, Martinsried, Germany. jwisniew@biochem.mpg.de
Zougman
Alexandre
A
Krüger
Sonja
S
Ziółkowski
Piotr
P
Pudełko
Marek
M
Bebenek
Marek
M
Mann
Matthias
M
D000107:Acetylation; D000595:Amino Acid Sequence; D001665:Binding Sites; D001943:Breast Neoplasms / Q000378:metabolism; D002455:Cell Division; D004247:DNA / Q000378:metabolism; D018983:DNA Footprinting; D016195:G2 Phase; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D040901:Proteomics
C426654:NUCKS1 protein, human; D009687:Nuclear Proteins; D010455:Peptides; D010750:Phosphoproteins; D004247:DNA; D008239:Lysine