HEAT-STABLE INHIBITORS OF CAMP-DEPENDENT PROTEIN-KINASE CARRY A NUCLEAR EXPORT SIGNAL

The heat-stable inhibitor of cAMP-dependent protein kinase (PKI) was s hown previously to export the kinase catalytic subunit (C) from the nu cleus (Fantozzi, D. A., Harootunian, A. T., Wen, W., Taylor, S. S., Fe ramisco, J. R., Tsien, R. Y., and Meinkoth, J. L. (1994) J. Biol. Chem . 269, 2676-2686), in addition to its ability to inhibit kinase activi ty. In this study, the mechanism of PKI export is investigated. The in jection of a C PHI complex containing both labeled PHI and C-subunit r evealed that both proteins exit the nucleus in unison. A fusion protei n of C-subunit with glutathione S-transferase (GST) (140 kDa) cannot t raverse the nuclear membrane in either direction, but can be exported from the nucleus when complexed with PHI, supporting the presence of a nuclear export signal (NES) in the C.PKI complex. Fusions of PKI alph a with GST (70 kDa) or PKI beta 1 with maltose-binding protein (MBP) ( 50 kDa) remain effective at exporting complexes with C-subunit. The ex port of C.PKI is also sensitive to temperature and energy depletion. T aken together, these results demonstrate that export is both energy- a nd temperature-dependent, but size-independent, consistent with an act ive signal-mediated export process. GST-PKI exits from the nucleus eve n in the absence of C-subunit, indicating that the NES resides entirel y on PKI, but suggesting that fusion of PKI to GST leads 60 a conforma tional change that mimics the exposure of the NES caused by the bindin g of C. Since both PKI alpha and PKI beta 1 can export C-subunit, the predicted export signal is likely to reside on the residues conserved between PKI alpha and PKI beta 1.