TI - betaarr2 interacts with 14-3-3 proteins and kinesin Kif3A . AB - 14-3-3 proteins ( comprising beta , gamma , epsilon , eta , zeta , tau and sigma isoforms ) are molecular adaptors , which often interact with consensus PHOSphorylated serine/threonine motifs of many proteins , thereby controlling a wide array of processes including signalling , cell cycle and apoptosis [29] . Interestingly , the 14-3-3zeta isoform was found to interact with the aPKC-Par3-Par6 polarity cassette [30] , whereas depletion of 14-3-3eta , which was found in a molecular complex with Par3 and the kinesin Kif3A , resulted in ciliogenesis defects [5] . Unpublished yeast two hybrid data revealed that betaarrs interact with 14-3-3 proteins , in agreement with a recent proteomic study [31] . The implication of 14-3-3eta in ciliogenesis and its connection with intraciliary transport through Kif3A , prompted us to characterize these interactions with betaarr2 . Endogenous 14-3-3 proteins were precipitated by a GST-betaarr2 fusion (Figure 7A) and the interaction between betaarr2 and the 14-3-3zeta isoform was confirmed by co-immunoprecipitation experiments showing that endogenous 14-3-3zeta interacts with Flag-tagged betaarr2 (Figure 7B) . The betaarr2 C-terminus contains a motif (RPQSAP) , similar to PHOSphorylated 14-3-3 consensus binding sites (Figure 7B) . However , mutation of Ser361 within this motif did not affect the interaction of betaarr2 with endogenous 14-3-3zeta , which co-immunoprecipitated as efficiently with both the S361A and S361D mutants of betaarr2 (Figure 7B) , indicating that the interaction of betaarr2 with 14-3-3 might be constitutive . This hypothesis is consistent with the observations that 14-3-3 proteins interact with recombinant GST-betaarr2 and that the 14-3-3/betaarrs interaction was not affected by GPCR activation ( data not shown and ref [31] ) . A PHOSphorylation -independent interaction of betaarr2 with 14-3-3 proteins would not be unique , since it has already been reported for other partners of 14-3-3 [29] . Interaction of betaarr2 with 14-3-3zeta was extended to the other isoforms and we found that flag-tagged betaarr2 , could co-immunoprecipitate with almost all 14-3-3 proteins ( data not shown ) , including 14-3-3eta (Figure 7C) , the isoform which has been implicated in ciliogenesis [5] . Finally , the possible colocalization of 14-3-3 proteins with betaarr2 was analyzed at the centrosome and PCs . In contrast to what was observed in kidney cells [5] , 14-3-3 proteins were not detected on the axoneme of PCs in RPE1 or MEFs . In these cells , 14-3-3 proteins were only found at the centrosome or basal body where they colocalized with gamma tubulin ( Figures S8 and data not shown ) and with betaarr2 ( Figure 7D and data not shown ) . Endogenous Kif3A was also precipitated by GST-betaarr2 (Figure 8A) , an interaction confirmed by colocalization studies . Indeed , as reported in vivo[32] , Kif3A was found in the cytoplasm and at the tip of the axoneme where it was colocalized with betaarr2 (Figure 8B) . Finally , because Kif3A was reported to co-immunoprecipitate with 14-3-3eta [5] , [30] , we investigated whether betaarr2 could be present in the same molecular complex . Supporting this hypothesis myc-14-3-3eta co-immunoprecipitated with both Flag-tagged betaarr2 and endogenous Kif3A (Figure 8C) .