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Phosphorylation and oligomerization states of native pig brain HSP90 studied by mass spectrometry

Garnier, Cyrille, Lafitte, Daniel, Jorgensen, Thomas J. D., Jensen, Ole N., Briand, Claudette, Peyrot, Vincent
European journal of biochemistry 2001 v.268 no.8 pp. 2402-2407
bioactive properties, biological speciation, brain, crosslinking, cytosol, electrospray ionization mass spectrometry, eukaryotic cells, intermediate filaments, phosphorylation, polyacrylamide gel electrophoresis, protein isoforms, protein kinases, receptors, signal transduction, swine
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 α‐HSP90 and β‐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 α isoform being ≈ six times more abundant than the β isoform. ESI‐MS in combination with λ phosphatase treatment provided direct evidence of the existence of four phosphorylated forms of native pig brain α‐HSP90, with the diphosphorylated form being the most abundant. For the β isoform, the di‐phosphorylated was also the most abundant. MALDI mass spectra of HSP90 samples after chemical cross‐linking showed a high percentage of α–α homodimers. In addition, evidence for the existence of higher HSP90 oligomers was obtained.