Endoplasmic reticulium protein profiling of heat-stressed Jurkat cells by one dimensional electrophoresis and liquid chromatography tandem mass spectrometry
- 127 Downloads
Proteomic study on membrane-integrated proteins in endoplasmic reticulum (ER) fractions was performed. In this study, we examined the effects of heat stress on Jurkat cells. The ER fractions were highly purified by differential centrifugation with sodium carbonate washing and acetone methanol precipitations. The ER membrane proteins were separated by one dimensional electrophoresis (1-DE), and some of the protein bands changed their abundance by heat stress, 12 of the 14 bands containing 40 and 60 ribosomal proteins whose expression level were decreased, on the contrary, 2 of the 14 bands containing ubiquitin and eukaryotic translation initiation factor 3 were increased. Heat treatment of human Jurkat cells led to an increase in the phosphorylation of PERK and eIF2α within 30 min of exposure. This was followed by an increase in the expression of the GRP78. Protein ubiquitination and subsequent degradation by the proteasome are important mechanisms regulating cell cycle, growth and differentiation, the result showed that heat stress enhanced ubiquitination modification of the microsomal proteins. The data of this study strongly suggest that heat treatment led to a significant reduction in protein expression and activated UPR, concomitant with protein hyperubiqutination in ER.
KeywordsEndoplasmic reticulium Heat stress SDS-PAGE LC–MS/MS
60S ribosomal protein L19
- CBB R-250
Coomassie brilliant blue R-250
Sodium lauryl sulfate
Polyacrylamide gel electrophoresis
Tris buffered saline
MPI preparation was supported by Prof. Bill Jordan (Center for Biodiscovery and School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand).
Conflict of interest
The authors declared that they have no competing interests.
- Fujimoto M, Nagasaka Y, Tanaka T, Nakamura K (1998) Analysis of heat shock-induced monophosphorylation of stathmin in human T lymphoblastic cell line JURKAT by two-dimensional gel electrophoresis. Electrophoresis 19:2515–2520Google Scholar
- Hetz C (2012) The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol 13:89–102Google Scholar
- Nakamura K, Zhang XL, Kuramitsu Y, Fujimoto M, Yuan XQ, Akada J, Aoshima-Okuda M, Mitani N, Itoh Y, Katoh T, Morita Y, Nagasaka Y, Yamazaki Y, Kuriki T, Sobel A (2006) Analysis on heat stress-induced hyperphosphorylation of stathmin at serine 37 in Jurkat cells by means of two-dimensional gel electrophoresis and tandem mass spectrometry. J Chromatogr A 1106:181–189CrossRefGoogle Scholar
- Nomura S, Kashiwagi S, Ito H, Mimura Y, Nakamura K (1993) Degradation of fibrinogen and fibrin by plasmin and nonplasmin proteases in the chronic subdural hematoma: evaluation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot. Electrophoresis 14:1318–1321CrossRefGoogle Scholar
- Saito A, Ochiai K, Kondo S, Tsumagari K, Murakami T, Cavener DR, Imaizumi K (2011) Endoplasmic reticulum stress response mediated by the PERK-eIF2α-ATF4 pathway is involved in osteoblast differentiation induced by BMP2. J Biol Chem 286(6):4809–4818Google Scholar
- Zhu G, Lee AS (2014) Role of the unfolded protein response, GRP78 and GRP94 in organ homeostasis. J Cell Physiol. doi: 10.1002/jcp.24923