P50(Cdc37) can buffer the temperature-sensitive properties of a mutant of Hck

Genetic studies have previously revealed that Cdc37p is required for the ca talytic competence of v-Src in yeast. We have reasoned that temperature-sen sitive mutants of Src family kinases might be more sensitive to the cellula r level of p50(Cdc37), the mammalian homolog of Cdc37p, than their wild-typ e counterpart, thus potentially providing a unique opportunity to elucidate the involvement of p50(Cdc37) in the folding and stabilization of Src fami ly kinases. A temperature-sensitive mutant of a constitutively active form of Hck (i.e., tsHck499F) was created by mutating two amino acids within the kinase domain of Hck499F. Significantly, overexpression of p50(Cdc37) resc ues the catalytic activity of tsHck499F at 33 degrees C, while partially bu ffering it against inactivation at higher temperatures (e.g., 37 and 39 deg rees C). Hsp90 function is required for tsHck499F activity and its stabiliz ation by p50(Cdc37), but overexpression of Hsp90 is not sufficient to stabi lize tsHck499F. Overexpression of p50(Cdc37) promotes the association of ts HcK499F with Hsp90, suggesting that the cellular level of p50Cdc37 might be the rate-limiting step in the association of tsHck499F with Hsp90. A trunc ation mutant of p50Cdc37 that cannot bind Hsp90 still has a limited capacit y to rescue the catalytic activity of tsHck499F and promote its association with Hsp90. This is a particularly important observation, since it argues that rather than solely acting as a passive adapter protein to tether tsHcK 499F to Hsp90, p50(Cdc37) may also act allosterically to enhance the associ ation of tsHcK499F with Hsp90, The findings presented here might also have implications for our understanding of the evolution of protein kinases and tumor development.