title: 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.
abstract: Two-dimensional gel electrophoresis (2-DE) and tandem mass spectrometry were successfully used for determination of a phosphorylation site of stathmin induced by heat stress to Jurkat cells of a human T lymphoblastic cell line. The cells were incubated for 30 min at 41 degrees C up to 45 degrees C in a serum free 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered culture medium. The intracellular soluble proteins were separated by 2-DE, and some of the proteins increased their abundance by heat stress. Those proteins were identified to be calmodulin, protein kinase C substrate, thymosin beta-4 and F-actin capping protein beta-subunit by peptide mass fingerprinting (PMF) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). On the contrary, protein phosphatase 2C gamma-isoform, nucleophosmin, translationally controlled tumor protein, Rho GDP-dissociation inhibitor-1, eukaryotic translation initiation factors 5A and 3A subunit 2, ubiquitin-like protein SMT 3B and chloride intracellular channel protein-1 were decreased their abundance. A protein spot of M(r) 18,000 and pI 5.9 was markedly increased at temperatures higher than 43 degrees C at which the cells were led to apoptosis. The spot was identified to be stathmin of a signal relay protein which has a function of sequestering microtubule. MALDI-quadrupole ion trap (QIT)-TOF-MS/MS and immunoblotting with a monoclonal antibody specific for a phosphorylation site of stathmin showed that the spot was a phosphorylated stathmin at serine 37 (Ser 37). The phosphorylation was suppressed by treatment of cells with olomoucine of an inhibitor specific for cyclin dependent kinase (Cdk-1). These results strongly suggest that heat stress activates Cdk-1 which phosphorylates Ser 37 on the stathmin molecule. The phosphorylation may cause the functional loss of stathmin for dynamic microtubule assembly and leads Jurkat cells to cell cycle arrest and apoptosis.
authors.affiliation: Department of Biochemistry and Biomolecular Recognition, Yamaguchi University School of Medicine, Minami-kogushi 1-1-1, Ube, Yamaguchi 755-8505, Japan. nakamura@yamaguchi-u.ac.jp
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Nakamura
authors.forename: Xiulian
authors.initials: X
authors.lastname: Zhang
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kuramitsu
authors.forename: Masanori
authors.initials: M
authors.lastname: Fujimoto
authors.forename: Xiaoqin
authors.initials: X
authors.lastname: Yuan
authors.forename: Junko
authors.initials: J
authors.lastname: Akada
authors.forename: Mikako
authors.initials: M
authors.lastname: Aoshima-Okuda
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Mitani
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Itoh
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Katoh
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Morita
authors.forename: Yuji
authors.initials: Y
authors.lastname: Nagasaka
authors.forename: Yuzo
authors.initials: Y
authors.lastname: Yamazaki
authors.forename: Tomoko
authors.initials: T
authors.lastname: Kuriki
authors.forename: Andre
authors.initials: A
authors.lastname: Sobel
mesh_terms: D000595:Amino Acid Sequence; D017209:Apoptosis; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D018882:Heat Stress Disorders / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D050777:Stathmin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D050777:Stathmin; D012694:Serine