The 100 kDa protein was identified as Grp94 by immunoprecipitation and reconstitution experiments. The mobility of the 100 kDa phosphoprotein was indistinguishable with that of an endoplasmic reticulum (ER)-resident molecular chaperone glucose-regulated protein 94 (Grp94) belonging to the Hsp90 family, and purified Grp94 was phosphorylated by a kinase in cell lysates in an NLS-dependent fashion. The mutated NLS-peptide (CPK TKRKVEDP) and the reversed NLS-peptide (PDEVKRKKKPC) are weak in the nuclear localization activity, and they only weakly stimulated phosphorylation of these substrates. When crude cell lysates were incubated with ATP, phosphorylation of several endogenous substrates with molecular masses of 100, 80, 50, and 45 kDa by an endogenous kinase was stimulated by the addition of the wild type NLS-peptide (CPKKKRKVEDP). In this study, we investigated effects of the NLS-peptide of Large T antigen on protein phosphorylation. Phosphorylation often modulates the intracellular distribution of signaling proteins. The nuclear localization signal sequence (NLS) of SV40 Large T antigen is essential and sufficient for the nuclear translocation of the protein.
