Two human 90-kDa heat shock proteins are phosphorylated in vivo at conserved serines that are phosphorylated in vitro by casein kinase II.

Amino-terminal protein sequence analysis revealed that exponentially growing human HeLa cells at 37 degrees C express two closely related 90-kDa "heat shock" proteins (hsp 90) in nearly equal amounts. Both hsp 90s begin with proline; the initial methionine residue is removed. The alpha protein contains a 9-amino acid segment, TQTQDQPME, from residues 4 to 12, that is replaced by a 4-amino acid segment, VHHG, in the beta form. The purified hsp 90 mixture contains 2 mol of phosphate/mol of polypeptide. Both hsp 90 proteins are phosphorylated at two homologous sites. For the alpha protein, these sites correspond to serine 231 and serine 263. A 5-amino acid segment, ESEDK, between the two phosphorylation sites is absent from the beta protein. The sequence between phosphorylation sites of both hsp 90s is predicted to have alpha helical structure. Dephosphorylated hsp 90 is phosphorylated at both sites by casein kinase II from HeLa cells, calf thymus, or rabbit reticulocytes; no other hsp 90 residues were phosphorylated by casein kinase II in vitro.