Figure 4. Co-immunoprecipitation of PDE4A5 and mutant species with MK2.

PDE4A5-binding sites for MK2 interaction were mutated as indicated and expressed in COS-1 cells. Immunoprecipitations of endogenous MK2 were prepared from cellular lysates and the co-purification of PDE4A5 and PDE4A5 mutants was assessed by western blotting using PDE4A5-specific antibody. The specific PDE4A5 mutants tested were (a) the FLY motif, (b) individual amino acids within the FLY motif and (c) other motifs that had been identified as being important for the MK2 association (Figure 3). Quantification of the importance of the FLY (Phe141, Leu142, Tyr143 cluster), ILD (Ile654, Leu655, Asp656 cluster), WHYS (Trp664, Tyr665, His666, Ser667 cluster), FQF (Phe693, Gln694, Phe695 cluster) and TLEE (Thr698, Leu699, Glu700, Glu701 cluster) motifs are shown in (d). Significances of mutations evaluated using Student's t-test compared with wt PDE4A5. P < 0.05 (*), P < 0.01 (**), P < 0.01 (***).