FIGURE 4.

RSPH3 phosphorylation dependent on the ERK1/2 pathway. A, HEK293 expressing 3xFLAG-RSP3H wild-type (wt) or T286A were placed in serum-free medium for 20 h and then labeled with 1 mCi/ml [³²P]orthophosphate with 10 μm U0126 or diluent for 60 min, and then the indicated groups were treated with 10 ng/ml EGF for 10 min. Autoradiograms of the 3xFLAG-RSP3H immunoprecipitates are shown. n = 3. IB, immunoblot; WCL, whole cell lysate. B, quantification of ³²P incorporation into the RSP3H bands from one experiment. Incorporation was normalized to the U0126-treated RSPH3 wild-type sample and adjusted for differences in protein expression as assessed by immunoblotting and densitometry. C and D, 3xFLAG-RSP3H wild-type, T286A, or T243V/T286A was immunoprecipitated from HEK293 cells and used as substrate for in vitro kinase assays with pERK1. Graph represents fold ³²P incorporation into RSP3H wild-type compared with the mutants (mean + S.E.). Protein amount in the immunoprecipitates was measured via densitometric analysis. Incorporation was normalized to that into the wild-type protein and corrected for differences in protein amount. n = 3. Mean values for T286A and T243V/T286A RSPH3 were 0.32 and 0.21, respectively. Statistical significance was evaluated with a nonparametric Kruskal-Wallis analysis.