Skip to main content
. 2015 Oct 12;6(35):37792–37807. doi: 10.18632/oncotarget.6096

Figure 4. Abi1-Iso2 competes with Crk for Abl kinase Proline-rich domain.

Figure 4

In vitro kinase assay performed with bacterially purified WtCrk and Abi1-Iso2 and Abl-1b purified from SF9 cells. 10nM Abl-1b was incubated with 1uM Crk alone or in presence of increase concentrations of Abi1-Iso2 with ATP at 25°C for 10mins. A. Crk mediated Abl transactivation was significantly suppressed in samples containing Abi1-Iso2 as shown by Abl pY245 blots. B. In another variation, Abi1 alone downregulates Abl activation alone and in presence of Crk in dose- dependent manner as indicated by Abl pY412 blot. C.-D. In vitro kinase assay to assess Abi1 SH3 domain on Abl kinase activity: Abl-1b and Crk were co-incubated with either C. Wt SH3 domain or D. mutant SH3 domain of Abi1-Iso2 in increasing amounts. In a dose-dependent manner Abi1 Wt-SH3 domain significantly suppresses Abl-1b-mediated CrkY221 phosphorylation (C), whereas mutant SH3 domain looses the capacity to suppress aforementioned CrkY221 phosphorylation (D). E. Competitive kinase assay: Abl kinase was co-incubated either with Crk alone or Crk and 2μM Abi1-Iso2 SH3 domain in kinase buffer. After 30min, Abl kinase activity was assessed by CrkY221 phosphorylation. Data are represented as mean ± SEM and n = 3 (P < 0.05).