Threonine 133 in RasGRP3 is phosphorylated in vitro by PKCθ. (A) MS/MS spectrum of phosphopeptide m/z 739.36 (131-RVpTQR-135) derived from analysis of RasGRP3 that was subjected to phosphorylation by recombinant PKCθ. The fragment ions are labeled with peak assignments (a, b, or y ions) conforming to the common notation used for peptide fragment ions. The peak labeled MH+ corresponds to the protonated ion of the phosphorylated parental tryptic peptide. Peaks resulting from neutral loss of a phosphate group from an ion are marked “-P.” * denotes the loss of an “-NH3”; Δ, the loss of a “-C(NH)2”; and r, immonium ion of arginine. (B) Generic (top) and specific (bottom) structural representations of a, b, and y fragment ions are shown. (C) A schematic diagram of RasGRP3 with the position of the deduced phosphorylation and the sequence of the diagnostic peptide shown. REM indicates Ras exchange motif; CDC25, Ras activator domain; EF, calcium-binding domain; and C1, DAG-binding domain. (D) The region surrounding the PKC phosphorylation site in human RasGRP3 is shown aligned with other RasGRP family members including mouse and rat RasGRP1. cRasGRP indicates C elegans RasGRP. Consensus sequence is summarized at the bottom (consensus). The position of the partially conserved Thr is shown with a vertical arrow, while the box highlights flanking basic residues. The amino terminal region of the CDC25 box is also indicated. Conserved sequences are indicated by shading.
