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

Abstract

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 a-HSP90 and b-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 a isoform being < six times more abundant than the b isoform. ESI-MS in combination with l phosphatase treatment provided direct evidence of the existence of four phosphorylated forms of native pig brain a-HSP90, with the diphosphorylated form being the most abundant. For the b isoform, the di-phosphorylated was also the most abundant. MALDI mass spectra of HSP90 samples after chemical cross-linking showed a high percentage of a±a homodimers. In addition, evidence for the existence of higher HSP90 oligomers was obtained.Keywords: HSP90; mass spectrometry; phosphorylation; cross-linking.Heat shock proteins (HSP) are abundant eukaryotic proteins constituting < 1 or 2% of the cellular proteins under physiological conditions, with an increased expression in response to stress [1]. These ubiquitous proteins, classified according to their apparent molecular mass, i.e. HSP27, 60, 70 or 90, work in close association with cytosolic cofactors to help the correct folding of several cytosolic proteins (reviewed in [2]).The target of this study is HSP90, a dimeric phosphoprotein involved in many cell processes such as cell cycle control, transcriptional regulation or signal transduction (reviewed in [3]). Mammalian HSP90 exists in two isoforms a-and b-HSP90 [4], differing in mass by < 2 kDa. Although dimers appear to be the functional form of HSP90, oligomeric forms exist in solution under physiological conditions [5] and their amount tends to increase under hyperthermal exposure [6±9]. HSP90 possesses an ATP binding site [10,11] and undergoes conformational changes in presence of ATP [12,13]. This protein also possesses ATPase activity [14]. The functions of HSP90 are in part regulated by phosphorylation, e.g. phosphorylated HSP90 stimulates eIF-2a kinase activity [15] and phosphorylation regulates the interaction between HSP90 and pp60v-src [16]. HSP phosphorylation may be mediated by various kinases [17,18] but this protein can also autophosphorylate and act as a kinase for other protein substrates [19].ESI-MS and MALDI-MS are the most suitable mass spectrometric techniques for the analysis of macromolecules [20,21]. The increase in resolution achieved by the latest generation of mass spectrometers allows a detailed investigation of post-translational modifications of large proteins and protein complexes [22]. This is particularly important ...

Binding of ATP to Heat Shock Protein 90

“…Vertebrates hsp90␣ and hsp90␤ are related by 85% amino acid identity in the same species, and all hsp90␣ or hsp90␤ show more than 95% identity between them (96% for chicken and human). Thus, results obtained for native pig hsp90, composed of 83% ␣-isoform and 17% ␤-isoform (20), recombinant ␣-chicken domains, and ␣-or ␤-human (predictions) are highly comparable and could be applied to ␣-or ␤-hsp90 from other eukaryotic species. This was confirmed by investigation of hsp90 secondary structure by spectral (CD and FTIR) and by predictive methods (PHD and nnpredict).…”

“…18, modified by Garnier et al (19,20). N-hsp90 (positions 1-221) and C-hsp90 domains were obtained by PCR amplification using a chicken hsp90 cDNA-bearing plasmid pSKB3 90 (13) and…”

“…18, modified by Garnier et al (19,20). N-hsp90 (positions 1-221) and C-hsp90 domains were obtained by PCR amplification using a chicken hsp90 cDNA-bearing plasmid pSKB3 90 (13) and * This work was supported by CNRS, "Association pour la Recherche contre le Cancer," and "ligue contre le cancer (indre et loire et île de France)" and International Association for the Promotion of Cooperation with Scientists from the New Independent States of the Former Soviet Union/Russian Foundation for Basic Research Grant 97-105.…”

Interactions between Human UGT1A1, UGT1A4, and UGT1A6 Affect Their Enzymatic Activities

“…In our recent study (Fujiwara et al, 2007), heterodimerization was detected with immunoprecipitation and thermal stability assays. In the present study, we performed native PAGE analyses to detect homodimerization and heterodimerization of UGTs, because accumulating data demonstrated the usefulness of this method to detect homo-and hetero-oligomeric complexes of protein (Garnier et al, 2001;Krause et al, 2004;Strohmeier et al, 2006). In all cases of single expression systems of UGT1A1, UGT1A4, and UGT1A6, bands corresponding to homodimers were observed (Fig.…”

Hsp90 phosphorylation, Wee1 and the cell cycle

“…[27][28][29][30][31][32][33][34][35][36][37][38][39] However our understanding of the role played by phosphorylation of distinct residues in regulating the chaperone function of Hsp90 remains incomplete. A number of serine and threonine phosphorylation sites …”