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 cel ls. HSP90 forms transient or stable complexes with several key proteins inv olved in signal transduction including protooncogenic protein kinases and n uclear receptors, it interacts with cellular structural elements such as ac tin-microfilament, tubulin-microtubule and intermediate filaments, and also exhibits conventional chaperone functions. This protein exists in two isof orms alpha -HSP90 and beta -HSP90, and it forms dimers which are crucial sp ecies for its biological activity. PAGE, ESI-MS and MALDI-MS were used to s tudy HSP90 purified from pig brain. The two protein isoforms were clearly d istinguished by ESI-MS, the alpha isoform being approximate to six times mo re abundant than the beta isoform. ESI-MS in combination with lambda phosph atase treatment provided direct evidence of the existence of four phosphory lated forms of native pig brain alpha -HSP90, with the diphosphorylated for m 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 additi on, evidence for the existence of higher HSP90 oligomers was obtained.