MULTIPLE INDEPENDENT INPUTS ARE REQUIRED FOR ACTIVATION OF THE P70 S6KINASE

Previous studies have shown that the noncatalytic carboxy-terminal tai l of the p70 S6 kinase (amino acids 422 to 525) contains an autoinhibi tory pseudosubstrate domain that is phosphorylated in situ during acti vation and in vitro by mitogen-activated protein kinases, The present study shows that a recombinant p70 deleted of the carboxy-terminal tai l (p70 Delta CT104) nevertheless exhibits a basal and serum-stimulated 40S kinase activity and susceptibility to inhibition by wortmannin ve ry similar to those of the parent, full-length p70 kinase, Carboxy-ter minal deletion reduces the extent of maximal inhibition produced by ra pamycin, from >95% in the full-length p70 to 60 to 80% in p70 Delta CT 104, without altering the sensitivity to rapamycin inhibition (50% inh ibitory concentration of 2 nM), Serum activation of p70 Delta CT104, a s with the parent, full-length p70, is accompanied by an increase in P -32 content (about twofold) in situ and a slowing in electrophoretic m obility; both modifications are inhibited by pretreatment with wortman nin or rapamycin, P-32-peptide maps of p70 Delta CT104 show multisite phosphorylation, and wortmannin and rapamycin appear to cause preferen tial dephosphorylation of the same subset of sites, Thus, it is likely that activation of the kinase requires phosphorylation of p70 at site s in addition to those previously identified in the carboxy-terminal t ail. Evidence that the carboxy-terminal tail actually functions as a p otent intramolecular inhibitor of kinase activity in situ is uncovered by deletion of a short acidic segment (amino acids 29 to 46) from the p70 amino-terminal noncatalytic region, Deletion of amino acids 29 to 46 causes a >95% inhibition of p70 activity despite continued phospho rylation of the carboxy-terminal tail in situ; additional deletion of the carboxy-terminal tail (yielding p70 Delta 29-46/Delta CT104) incre ases activity 10 fold, to a level approaching that of p70 Delta CT101. Deletion of residues 29 to 46 also abolishes completely the sensitivi ty of p70 to inhibition by rapamycin but does not alter the susceptibi lity to activation by serum or inhibition by wortmannin. Although the mechanisms underlying the effects of the Delta 29-46 deletion are not known, they are not attributable to loss of the major in situ p70 phos phorylation site at Ser-40, Thus, activation of the p70 S6 kinase invo lves multiple, independent inputs directed at different domains of the p70 polypeptide, Disinhibition from the carboxy-terminal tail require s, in addition to its multisite phosphorylation, an activating input d ependent on the presence of amino acids 29 to 46; this p70-activating input may be the same as that inhibited by rapamycin but is distinct f rom that arising from the wortmannin-inhibitable phosphatidylinositol 3-kinase, In addition, as exemplified by the rapamycin-resistant but m itogen- and wortmannin-sensitive p70 Delta 29-46/Delta CT104 mutant, a further activating input, which probably involves site-specific phosp horylation in the segment between amino acids 46 to 421, is necessary.