THE IMMUNOGLOBULIN-BINDING PROTEIN INVITRO AUTOPHOSPHORYLATION SITE MAPS TO A THREONINE WITHIN THE ATP BINDING CLEFT BUT IS NOT A DETECTABLE SITE OF INVIVO PHOSPHORYLATION

In vitro incubation of immunoprecipitated immunoglobulin-binding prote in (BiP) complexes with calcium and [gamma-P-32]ATP resulted in the ph osphorylation of BiP on a threonine residue. This autophosphorylation activity did not occur in the presence of magnesium but had the same p H optimum as reported for its magnesium-dependent ATPase activity. Thi s suggested the possibility that both activities could occur through A TP hydrolysis at the same site. In support of this, mutation of either Thr-37 or Thr-229 to a glycine eliminated both autophosphorylation an d ATPase activities, and mutation of either residue to a serine signif icantly reduced both activities. Glutamic acid 175 in HSC71 has been h ypothesized to flank the divalent cation complexed with ATP. Mutation of the analogous glutamic acid, Glu-201, in BiP abolished ATPase activ ity but still supported some autophosphorylation. The in vitro phospho rylation site was mapped to Thr-229 by mutational analysis. This threo nine has been hypothesized to interact with the gamma-phosphate of ATP through a polarized water molecule and would be in a position to act as a phosphate acceptor in the ATP hydrolysis reaction. These data imp ly that both ATPase and autophosphorylation result from ATP hydrolysis at the same site and that the cation associated with BiP determines w hich activity is observed. Comparison of partial protease digestion or cyanogen bromide cleavage products of in vitro and in vivo phosphoryl ated BiP demonstrated that Thr-229 is not a detectable site of phospho rylation in cells. Therefore, whatever functional role phosphorylation may have in vivo, it cannot be attributed to autophosphorylation of T hr-229.