Figure 4.

Ser-122 is required and sufficient for the phosphorylation of RACK1. A, Recombinant GST-tagged RACK1A and the point mutants were incubated with [γ-³²P]ATP and GST-WNK8 for 6 h at room temperature. Proteins were then separated by SDS-PAGE: S122D, RACK1A^S122D; T162E, RACK1A^T162E; and S122D/T162E, RACK1A^S122D/T162E. Note that the Asp substitution at Ser-122 induced a partial cleavage of RACK1A protein, which resulted in a cleaved protein that was approximately 5 kD smaller than the native RACK1A protein. Neither the intact protein nor the cleaved protein was phosphorylated by WNK8. B, Partial amino acid sequence indicating the positions of Ser-122 and Thr-162. Thr-161 of RACK1C corresponds to Thr-161 of RACK1B and Thr-162 of RACK1A.