Comprehensive characterization of heat shock protein 27 phosphorylation in human endothelial cells stimulated by the microbial dithiole thiolutin.
Thiolutin is a sulfur-based microbial compound with known activity as an angiogenesis inhibitor. Relative to previously studied angiogenesis inhibitors, thiolutin is a remarkably potent inducer of heat shock protein 27 (Hsp27) phosphorylation. This phosphorylation requires p38 kinase but is independent of increased p38 phosphorylation. To elucidate how thiolutin regulates Hsp27 phosphorylation and ultimately angiogenesis, Hsp27 was immunoprecipitated using nonphosphorylated and phospho-Ser78 specific antibodies from lysates of thiolutin treated and untreated human umbilical vein endothelial cells and analyzed by LC-MS. Separate LC-MS analyses of Lys-C, Lys-C plus trypsin, and Lys-C plus Glu-C digests provided 100% sequence coverage, including the identification of a very large 13 kDa Lys-C fragment using a special sample handling procedure (4 M guanidine HCl) prior to the LC-MS analysis to improve the large peptide recovery. The analysis revealed a novel post-translational modification of Hsp27 involving truncation of the N-terminal Met and acetylation of the penultimate Thr. Analysis of a Glu-C fragment containing two phosphorylation sites, Ser78 and Ser82, and a tryptic fragment containing the other phosphorylation site, Ser15, enabled quantitative stoichiometry of Hsp27 phosphorylation by LC-MS. The strategy revealed details of Hsp27 phosphorylation, including significant di-phosphorylation at both Ser78 and Ser82, that would be difficult to obtain by traditional approaches because oligomerization of the hydrophobic N-terminal region of the molecule prevents efficient enzymatic cleavage. The combination of Western blotting, immunoprecipation, and LC-MS provides a quantitative analysis of thiolutin-stimulated Hsp27 phosphorylation and further defines the role of Hsp27 in the antiangiogenic activities of thiolutin and related dithiolethiones.
Dai
Shujia
S
Barnett Institute, Northeastern University, Boston, Massachusetts 02115, USA.
Jia
Yifeng
Y
Wu
Shiaw-Lin
SL
Isenberg
Jeff S
JS
Ridnour
Lisa A
LA
Bandle
Russell W
RW
Wink
David A
DA
Roberts
David D
DD
Karger
Barry L
BL
D000595:Amino Acid Sequence; D020533:Angiogenesis Inhibitors / Q000494:pharmacology; D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D004789:Enzyme Activation; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010766:Phosphorylation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011760:Pyrrolidinones / Q000494:pharmacology; D017550:Spectroscopy, Fourier Transform Infrared; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D020533:Angiogenesis Inhibitors; D006360:Heat-Shock Proteins; D020033:Protein Isoforms; D011760:Pyrrolidinones; C006361:acetopyrrothine; D048051:p38 Mitogen-Activated Protein Kinases