Figure 5: Association of Monosiga brevicollis crka1 Protein with Mammalian Bcar1 and Rapgef1.

M. brevicollis crka1 associated with an HA-tagged mouse Bcar1 (HA-Mm Bcar1) when an activated mouse Src (Y529F) was co-expressed in a heterologous system (lane 2). Human embryonic kidney 293 cells (HEK293) were transfected with combinations of plasmids as indicated. M. brevicollis crka1 and human CRK were fused to an enhanced GFP at their N-terminus to enable immunoprecipitation (IP) with anti-GFP antibodies. Lanes 1 and 3 were samples from control groups without active Src, showing little co-precipitation of HA-Bcar1. Lane 4 was a positive control with human CRK instead of Mb crka1. Lanes 5 and 6 were negative controls in which GFP without human CRK or Mb crka1 SH2 domain was expressed. The amount of Bcar1 co-precipitation could not be directly compared between Mb crka1 and Hs CRK, since Hs CRK appeared to reduce protein levels of transfected HA-Mm Bcar1 and Src (reproducible in three experiments; see Western blots of cell lysates for HA and Src). Mb crka1 binds to multiple proteins phosphorylated by active Src. Phosphotyrosine contents were probed by an anti-phosphotyrosine antibody in the same immunoprecipitates used in panel a. While Bcar1 was the major protein that bound to Mb crka1 and Hs CRK (lanes 2 and 4), additional tyrosine-phosphorylated proteins of unknown identities (*) also associated with Mb crka1 (lane 2). We noted that GFP-fusion proteins and GFP itself were also phosphorylated by active Src (lanes 2, 4, and 6). (a) Monosiga brevicollis crka1 can associate with the CRK SH3 binding protein RAPGEF1 (also known as C3G), albeit at a lower level than that of human CRK (lanes 2 and 4). A heterologous system was designed to co-express RAPGEF1 with GFP-tagged Mb crka1 or Hs CRK in HEK293 cells as above.