70-KDA HEAT-SHOCK COGNATE PROTEIN INTERACTS DIRECTLY WITH THE N-TERMINAL REGION OF THE RETINOBLASTOMA GENE-PRODUCT PRB - IDENTIFICATION OF A NOVEL REGION OF PRB-MEDIATING PROTEIN-INTERACTION

Retinoblastoma protein (pRb) functions as a tumor suppressor, and cert ain proteins are known to bind to pRb in the C-terminal region. Althou gh the N-terminal region of pRb may also mediate interaction with some proteins, no such protein has been identified yet. We demonstrated pr eviously the in vivo protein association between pRb and 73-kDa heat s hock cognate protein (hsc73) in certain human tumor cell lines. In thi s report we analyzed the interaction between these two proteins in vit ro, Our data showed that hsc73 interacts with the novel N-terminal reg ion of pRb; that is, pRb binds directly to hsc73 and dissociates from hsc73 in an ATP-dependent manner. By using deletion mutants of cDNA en coding pRb, the hsc73 binding site of pRb was determined to be located in the region (residues 301-372) outside the so-called A pocket (resi dues 373-579) of this tumor suppressor protein. This finding was compa tible with the fact that the adenovirus E1A oncoprotein, which is know n to bind to the E2F binding pocket region of pRb, could not compete w ith hsc73 for the binding, Furthermore, phosphorylation of pRb by cycl in-dependent kinase inhibited the binding of pRb to hsc73. These data suggest that hsc73 may act exclusively as the molecular chaperone for nonphosphorylated pRb. As a result, hsc73 may function as a molecular stabilizer of nonphosphorylated pRb.