Figure 1. c-Abl Mediated Tyrosine Phosphorylation of hAha1.
(A) HEK293 cells were transiently transfected with empty plasmid (C) or hAha1-FLAG construct. The hAha1-FLAG was immunoprecipitated (IP) and tyrosine phosphorylation was detected by immunoblotting with pan anti-phospho-tyrosine antibody (4G10).
(B) Schematic representation of hAha1 showing all tyrosine residues and also the putative c-Abl targeted tyrosine consensus motif. The amino acid residues 162-201 correspond to linker (L) region.
(C) hAha1 tyrosine residues were mutated individually to phenylalanine (F), expressed, and purified from bacteria. The active c-Abl-GST was used to phosphorylate hAha1 wild-type (WT) and its non-phosphorylatable mutants in vitro. The tyrosine phosphorylation was detected by immunoblotting with pan anti-phospho-tyrosine antibody (4G10).
(D) HEK293 cells were transiently transfected with c-Abl and hAha1-FLAG (WT) or the non-phosphorylatable Y223F mutant. The tyrosine phosphorylation of IP hAha1-FLAG was detected by immunoblotting with pan anti-phospho-tyrosine antibody (4G10).
(E) hAha1-FLAG (WT) or the non-phosphorylatable Y223F mutant constructs were used to transiently transfect HEK293 cells for 24 hr. The cells were then treated with 20 μM DPH for 6 hr prior to lysis. The tyrosine phosphorylation of IP hAha1-FLAG was detected by immunoblotting with pan anti-phospho-tyrosine antibody (4G10).
(F) c-Abl deficient (c-Abl−/−) MEF cell line and the WT MEF cell line (c-Abl+/+) were transiently transfected with hAha1-FLAG (WT) or the non-phosphorylatable Y223F mutant. The IP hAha1-FLAG was immunoblotted and tyrosine phosphorylation was detected with pan anti-phospho-tyrosine antibody (4G10).
(G) WT yeast cells co-expressing GAL1-cABL1 and hAHA1-FLAG or Y223F mutant under yAHA1 native promoter were grown on either raffinose (–) or galactose (+). The tyrosine phosphorylation of hAha1 was assessed by IP and immunoblotting.
(H) In vitro phosphorylation of hAha1-Y223 by a purified and active c-Abl. The hAha1-FLAG or hAha1-Y223F-FLAG were bound to FLAG-beads and phosphorylated by active c-Abl-GST. The reaction was also carried out with the addition of hHsp90α-His6. The dephosphorylation of hAha1-Y223 was performed by addition of CIAP.