Hydrophobic Motif Phosphorylation Is Not Required for Activation Loop Phosphorylation of p70 Ribosomal Protein

p70 ribosomal protein S6 kinase 1 (S6K1) is regulated bymul-tiple phosphorylation events. Three of these sites are highly con-served among AGC kinases (cAMP dependent Protein Kinase, cGMPdependent ProteinKinase, andProteinKinaseC subfam-ily): the activation loop in the kinase domain, and two C-termi-nal sites, the turn motif and the hydrophobic motif. The com-mon dogma has been that phosphorylation of the hydrophobic motif primes S6K1 for the phosphorylation at the activation loop by phosphoinositide-dependent protein kinase 1 (PDK1). Here, we show that the turn motif is, in fact, phosphorylated first, the activation loop second, and the hydrophobic motif is third. Specifically, biochemical analyses of a construct of S6K1 lacking the C-terminal autoinhibitory domain as well as full-length S6K1, reveals that S6K1 is constitutively phosphorylated at the turn motif when expressed in insect cells and become