Nuclear translocation of the Hsp70/Hsp90 organizing protein mSTI1 is regulated by cell cycle kinases

Longshaw, V.M. and Chapple, J.P. and Cheetham, M.E. and Blatch, G.L. (2004) Nuclear translocation of the Hsp70/Hsp90 organizing protein mSTI1 is regulated by cell cycle kinases. Journal of Cell Science, 117 (5). pp. 701-710. ISSN 0021-9533



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The co-chaperone murine stress-inducible protein 1 (mSTI1), an Hsp70/Hsp90 organizing protein (Hop) homologue, mediates the assembly of the Hsp70/Hsp90 chaperone heterocomplex. The mSTI1 protein can be phosphorylated in vitro by cell cycle kinases proximal to a putative nuclear localization signal (NLS), which substantiated a predicted casein kinase II (CKII)-cdc2 kinase-NLS (CcN) motif at position 180-239 and suggested that mSTI1 might move between the cytoplasm and the nucleus under certain cell cycle conditions. The mechanism responsible for the cellular localization of mSTI1 was probed using NIH3T3 fibroblasts to investigate the localization of endogenous mSTI1 and enhanced green fluorescent protein (EGFP)-tagged mSTI1 mutants. Localization studies on cell lines stably expressing NLS(mSTI1)-EGFP and EGFP demonstrated that the NLS(mSTI1) was able to promote a nuclear localization of EGFP. The mSTI1 protein was exclusively cytoplasmic in most cells under normal conditions but was present in the nucleus of a subpopulation of cells and accumulated in the nucleus following inhibition of nuclear export (leptomycin B treatment). G1/S-phase arrest (using hydroxyurea) and inhibition of cdc2 kinase (using olomoucine) but not inhibition of casein kinase II (using 5,6-dichlorobenzimidazole riboside), increased the proportion of cells with endogenous mSTI1 nuclear staining. mSTI1-EGFP behaved identically to endogenous mSTI1. The functional importance of key residues was tested using modified mSTI1-EGFP proteins. Inactivation and phosphorylation mimicking of potential phosphorylation sites in mSTI1 altered the nuclear translocation. Mimicking of phosphorylation at the mSTI1 CKII phosphorylation site (S189E) promoted nuclear localization of mSTI1-EGFP. Mimicking phosphorylation at the cdc2 kinase phosphorylation site (T198E) promoted cytoplasmic localization of mSTI1-EGFP at the G1/S-phase transition,whereas removal of this site (T198A) promoted the nuclear localization of mSTI1-EGFP under the same conditions. These data provide the first evidence of nuclear import and export of a major Hsp70/Hsp90 co-chaperone and the regulation of this nuclear-cytoplasmic shuttling by cell cycle status and cell cycle kinases.

Item Type:Article
Additional Information:Research article
Uncontrolled Keywords:Sti1; Hop, Hsp70; Hsp90; Cdc2; CKII; adaptor protein; casein kinase II; green fluorescent protein; heat shock protein 70; heat shock protein 90; murine stress inducible protein 1; mutant protein; protein kinase; animal cell; cell cycle G1 phase; cell cycle S phase; cell line; cell nucleus; cell subpopulation; gene translocation; nuclear localization signal; protein expression; protein localization; protein motif; protein phosphorylation; Active Transport, Cell Nucleus; Casein Kinase II; CDC2 Protein Kinase; Cell Nucleus; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; NIH 3T3 Cells; Nuclear Localization Signals; Phosphoproteins; Protein Binding; Recombinant Fusion Proteins
Subjects:Y Unknown > Subjects to be assigned
Divisions:Faculty > Faculty of Science > Biochemistry, Microbiology & Biotechnology
ID Code:1398
Deposited By: Mrs Eileen Shepherd
Deposited On:10 Jun 2009
Last Modified:06 Jan 2012 16:20
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