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A Brassica napus cDNA encoding the 90 kDa heat shock protein, hsp90, was modified to add 6 histidines at the C-terminus and expressed in insect cells to prepare a recombinant histidine-tagged hsp90. The recombinant protein was purified over Ni2+-NTA agarose columns and its identity was confirmed by Western blotting, using a plant hsp90-specific antiserum. Incubation of purified hsp90 with [gamma-32P] ATP in the presence of Mn2+ resulted in its autophosphorylation on serine residues. The purified hsp90 could also phosphorylate other protein substrates such as histones and casein in the presence of Mn2+. Analysis of phosphorylated casein revealed that serine residues are phosphorylated by hsp90. This is the first demonstration that a cytosolic hsp90 homolog can phosphorylate other protein substrates.