Phosphorylation and oligomerization states of native pig brain HSP90 studied by mass spectrometry.
HSP90 is one of the most abundant proteins in the cytosol of eukaryotic cells. HSP90 forms transient or stable complexes with several key proteins involved in signal transduction including protooncogenic protein kinases and nuclear receptors, it interacts with cellular structural elements such as actin-microfilament, tubulin-microtubule and intermediate filaments, and also exhibits conventional chaperone functions. This protein exists in two isoforms alpha-HSP90 and beta-HSP90, and it forms dimers which are crucial species for its biological activity. PAGE, ESI-MS and MALDI-MS were used to study HSP90 purified from pig brain. The two protein isoforms were clearly distinguished by ESI-MS, the alpha isoform being approximately six times more abundant than the beta isoform. ESI-MS in combination with lambda phosphatase treatment provided direct evidence of the existence of four phosphorylated forms of native pig brain alpha-HSP90, with the diphosphorylated form being the most abundant. For the beta isoform, the di-phosphorylated was also the most abundant. MALDI mass spectra of HSP90 samples after chemical cross-linking showed a high percentage of alpha-alpha homodimers. In addition, evidence for the existence of higher HSP90 oligomers was obtained.
Garnier
C
C
UMR CNRS 6032, Faculté de Pharmacie, Marseille, France.
Lafitte
D
D
Jorgensen
T J
TJ
Jensen
O N
ON
Briand
C
C
Peyrot
V
V
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002845:Chromatography; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010744:Phosphoric Monoester Hydrolases / Q000494:pharmacology; D010766:Phosphorylation; D020033:Protein Isoforms; D011499:Protein Processing, Post-Translational; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine
D003432:Cross-Linking Reagents; D018841:HSP90 Heat-Shock Proteins; D020033:Protein Isoforms; D010744:Phosphoric Monoester Hydrolases