Characterization of heat shock protein 27 phosphorylation sites in renal cell carcinoma

Abstract

The phosphorylation of heat shock protein 27 (HSP27) occurs differently in human renal cell carcinoma (RCC) compared to homologous normal kidney tissue. Two-dimensional electrophoresis was used to separate and visualize HSP27, via immunostaining with anti-HSP27 antibody, in tumor and normal renal samples, obtained after surgery resection from patients with RCC. The mean number of protein species was 21 in RCC and 15 in normal tissues. Selected spots were in-gel digested with trypsin, extracted and analyzed by microcapillary liquid chromatography (LC) electrospray ionization tandem mass spectrometry to confirm HSP27 protein identity and reveal phosphorylation sites. Loss of phosphopeptides due to extensive plumbing and/or metal components in automated LC-systems was limited by manual loading of samples directly onto the LC system using a homemade pressure vessel. Mass spectrometry (MS) analysis revealed that in three of the HSP27 protein species phosphorylation occurred at Serine 15 and in five at Serine 82 in a different pattern. The phosphorylation of Serine 15 and 82 was also investigated by immunohistochemistry on tissue sections. The data obtained using anti-HSP27Serine82phos-antibody are consistent with MS results, while the variance between results achieved by anti-HSP27Serine15phos-antibody and by MS is probably due to the low specificity of the antibody. Knowledge of the diversity and modulation of HSP27 phosphorylation protein species might represent useful markers involved in the differentiation of RCC.

Heat shock protects cardiac cells from doxorubicin-induced toxicity by activating p38 MAPK and phosphorylation of small heat shock protein 27

“…Previous 2D gel electrophoresis studies of heart tissues and cell lines reported largely diversified results for the number of HSP27 spots. Depending on the cell type, the number of isoforms with different phosphorylation status varied from 2 to 80 (10,42,44,47). Especially in pathogenic tissues, increased numbers of HSP27 spots have been observed in 2D gels, likely because of disintegration of HSP27 oligomers and also different degrees of phosphorylation, such as, for example, mono-, di-, and triphosphorylation.…”

Role of heat shock factor-1 activation in the doxorubicin-induced heart failure in mice

“…Prominently, three spots were observed in control hearts. These three spots are assigned to nonphosphorylated, monophosphorylated, and diphosphorylated Hsp27 (5,29). In some studies Pro-Q staining (a marker of phosphorylated proteins) has stained all the three spots, and these studies have described this as mono-, di-, and triphosphorylated Hsp27 (8).…”

“…4). Based on previous studies of Hsp27, this m/z is for the nonphosphorylated peptide (5,29). Mass difference between COOH terminal containing fragments y6 and y7 as well as y7 and y8 is close to 87 (Fig.…”

Differential expression of alphaB-crystallin and Hsp27-1 in anaplastic thyroid carcinomas because of tumor-specific alphaB-crystallin gene (CRYAB) silencing

“…AlphaB-crystallin expression has also been demonstrated to varying extent in numerous epithelial tumors (Pinder et al 1994). Similarly, Hsp27-1 expression was found in various benign and malignant tissues, eg, in retinal neurons (Whitlock et al 2005a(Whitlock et al , 2005b, in reproductive tissues (White et al 2005), in normal and neoplastic renal tissue (Neuhofer et al 2005;Tremolada et al 2005), and in malignant thyroid tissue (Akslen and Varhaug 1995).…”