Mapping of autophosphorylation sites. (A) Identification of Y572, Y589, and Y591 as sites of in vivo autophosphorylation: PAE cells expressing WT or the indicated Y→F mutant of Flt3 were labeled with 2 to 6 mCi (74-222 MBq) [³²P]-orthophosphate for 6 hours before they were stimulated with FL for 10 minutes, and labeled Flt3 was immunoprecipitated (IP) and subsequently digested with trypsin. Tryptic fragments of Flt3 containing aa's 572-595 were precipitated with 1 μg of a peptide-specific antibody described in “In vivo ³²P- or- thophosphate labeling of cells” and subjected to phosphoamino acid analysis and Edman-based degradation (after an additional digest with AspN when indicated). The amount of radioactivity released in each cycle of Edman degradation was quantitated using a PhosphorImager and MultiGauge software (Fujifilm, Tokyo, Japan). (B) Identification of Y599 as in vivo autophosphorylation site: PAE cells expressing WT or Y599F-Flt3 were treated as described for panel A. The tryptic peptide containing aa's 596-602 of Flt3 was immunoprecipitated and processed as mentioned for panel A.