FIGURE 3.

The phosphorylation sites of GRK1. a, the RH-kinase core of GRK1. The structure corresponds to that of crystal form I (with composite C-terminal extension; see Fig. 2). The Ser⁵, Thr⁸, Ser²¹, Ser⁴⁸⁸, and Thr⁴⁸⁹ phosphorylation sites are drawn as stick models. The expected position of the membrane plane is indicated. Top inset, the Ser⁴⁸⁸ and Thr⁴⁸⁹ phosphorylation sites correspond to the AGC kinase turn motif. Bottom inset, interaction of Thr(P)⁸ with the RH domain. Gln⁷³ and Glu⁹³ form direct hydrogen bonds, whereas Lys⁶⁹ and Lys⁹⁰ complement the charge of the phosphate moiety. These crystals grew at pH 4.35, and so either Glu⁹³ or the phosphate group could be protonated. b, tandem mass spectrometry spectra of phosphopeptides from GRK1₅₃₅-His₆ (Pool A, pretreated with 4 mm ATP and 2 mm MgCl₂). Both Ser⁵ and Thr⁸ sites were identified in a single peptide. The Ser⁵ site was also readily observed in endogenous GRK1, as were the previously observed phosphorylation sites at Ser²¹, Ser⁴⁸⁸, and Thr⁴⁸⁹ (supplemental Fig. S7).