p70 S6 kinase sensitivity to rapamycin is eliminated by amino acid substitution of Thr229.
Rapamycin, which forms a complex with FK506-binding protein and FK506-binding protein-rapamycin-associated protein, induces immunosuppression through an as yet undefined pathway. Our previous studies demonstrated that rapamycin inactivates p7Os6k, which results in the inhibition of translation of ribosomal proteins. Here, we analyzed the mechanism of inactivation of p70s6k by rapamycin using site-directed mutagenesis of the phosphate acceptor site. We introduced a point mutation at Thr229 in the catalytic subdomain VIII of p7Os6k because Thr229 of p7Os6k corresponds to the phosphorylation site of mitogen-activated protein kinases by mitogen-activated protein kinase kinase and to the autophosphorylation site of protein kinase A whose phosphorylation is required for its full activation. Thr229 of rat p70s6k was substituted by either a neutral amino acid Ala (T229A) or by an acidic amino acid Glu (T229E). T229A-P70s6k, expressed in COS cells, migrated faster in SDS-polyacrylamide gels than wild-type p70s6k, and this mutation completely ablated the catalytic activity of the kinase. In contrast, T229E-p70s6k migrated more slowly in SDS-polyacrylamide gels, but demonstrated partial kinase activity (approximately 20% compared with the wild type). These data indicate that the negative charge at Thr229 which is normally achieved by phosphorylation of the residue, is important for the catalytic function of p70s6k. Further, the residual activity of T229E-p70s6k was not affected by rapamycin, implying that rapamycin-induced inactivation of p70s6k may be caused by dephosphorylation or impaired phosphorylation of Thr229.
Sugiyama
H
H
Division of Basic Sciences, Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO 80206, USA.
Papst
P
P
Gelfand
E W
EW
Terada
N
N
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017124:Conserved Sequence; D004351:Drug Resistance; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011090:Polyenes / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics; D051381:Rats; D019893:Ribosomal Protein S6 Kinases; D020123:Sirolimus; D013912:Threonine / Q000235:genetics* / Q000378:metabolism*
D011090:Polyenes; D013912:Threonine; D017346:Protein Serine-Threonine Kinases; D019893:Ribosomal Protein S6 Kinases; D020123:Sirolimus