PHYSIOLOGICAL INHIBITORS OF THE CATALYTIC SUBUNIT OF CAMP-DEPENDENT PROTEIN-KINASE - EFFECT OF MGATP ON PROTEIN-PROTEIN INTERACTIONS

The catalytic (C) subunit of cAMP-dependent protein kinase interacts w ith two classes of inhibitors. The regulatory (R) subunits, types I an d II, associate to form an inactive holoenzyme complex that is activat ed in response to cAMP. The C-subunit is also inhibited by small heat- stable protein kinase inhibitors (PKI's). Inhibition by both PKI and R (I)-subunit requires the synergistic high-affinity binding of MgATP. T he stabilizing effect of ATP was quantitated by using analytical gel c hromatography. Both the type I holoenzyme and the C.PKI complex in the presence of MgATP show apparent K(d)'s for subunit association that a re below 0.1 nM, while in the absence of MgATP the apparent K(d)'s are 125 nM and 2.3 muM, respectively, for the two complexes. In the absen ce of MgATP both complexes also can be dissociated readily and, hence, activated by salt-induced dissociation. Under physiological salt conc entrations, salt-induced dissociation would be substantial in the abse nce of the high-affinity binding of MgATP. In both complexes, the ATPa se activity of the free C-subunit is abolished. The off rates for MgAT P also indicate that the type I holoenzyme is more stable than the C-P KI complex. The off rate (t1/2) for MgATP from the C.PKI complex is 17 min, while the off rate for the type I holoenzyme is 11.7 h. When the C-PKI complex is incubated with R(I)-subunit in the presence or absen ce of MgATP, the C-subunit preferentially reassociates with the R(I)-s ubunit, forming holoenzyme. In contrast, free PKI cannot compete for t he C-subunit when it is part of a holoenzyme complex. These in vitro r esults suggest that the formation of the C.PKI complex may not be a de ad-end pathway. Instead, PKI could function as a shuttle mechanism for eventually returning the C-subunit to a holoenzyme complex.