Article Figures & Data
Figures
- Fig. 1.
In vitro binding of the Abi-1 SH3 domain to Sos. (A) MBP and MBP-Abi SH3 immobilized on amylose resin were incubated with BALB/c3T3 cell lysate. Bound proteins were immunoblotted with antibodies that recognize both Sos1 and Sos2 (αSos1/2) (top). The membrane was stripped and reprobed with anti-Sos1 (middle) and anti-Sos2 (bottom) antibodies. Total cell lysate and anti-Sos1 and -Sos2 immunoprecipitate (IP) from BALB/c3T3 cells were included for reference. WB, Western blot. (B) COS cells were transiently transfected with plasmids encoding HA-tagged DOKL, Abi(Δ1–85), or Sos2(874–1297) [Sos2(C)]. Cell lysates were incubated with MBP and MBP-Abi SH3 as described for panel A. Total cell lysates and bound proteins were probed with anti-HA antibody (upper). HA-Sos2(874–1297) expression in the total cell lysate was detected upon longer exposure. A fraction of the resin eluate was stained with Coomassie brilliant blue to confirm roughly equivalent amounts of MBP and MBP-Abi SH3 (lower).
- Fig. 2.
Interaction between Abi-1 and Sos in COS cells. (A) COS cells were transiently transfected with plasmids encoding myc-tagged GM-CSF receptor α chain, c-Abl, Abi-1, or AbiΔ328–356. Sos1 and Sos2 were immunoprecipitated from cell lysates. Precipitated proteins (top) were immunoblotted with anti-myc antibody (αmyc). The membrane from the top blot was reprobed with anti-Sos1 and -Sos2 antibodies to confirm equal levels of protein loading (middle). Total cell lysates were probed with anti-myc antibody to confirm expression of myc-tagged proteins (bottom). IP, immunoprecipitate; WB, Western blot. (B) Schematic representation of full-length (FL) Abi-1 and deletion mutant proteins used in panel C. The homeobox-like domain, proline-rich motifs, polyproline region, and SH3 domain are indicated. (C) COS cells were transiently transfected with plasmids encoding myc-tagged full-length Abi-1 or the indicated deletion mutant proteins. Proteins were immunoprecipitated from cell lysates with anti-Sos1 and -Sos2 antibodies and were analyzed as described for panel A. The asterisk indicates the position of the immunoglobulin heavy chain.
- Fig. 3.
Association of Abi-1 and Grb2 in COS cells. (A) COS cells were transiently cotransfected with the indicated HA-Sos1 and HA–Abi-1 plasmids. Grb2 was immunoprecipitated from cell lysates. Precipitated proteins (top) and total cell lysates (bottom) were immunoblotted with anti-HA antibody (αHA). The membrane from the top blot was reprobed with anti-Grb2 antibody to confirm equal levels of protein loading (middle). IP, immunoprecipitate; WB, Western blot. (B) COS cells were transiently cotransfected with the indicated HA-Sos1 and HA–Abi-1 plasmids. Serum-starved cells were left untreated or were stimulated with EGF (100 ng/ml) for 2 or 10 min. Cell lysates were prepared and incubated with anti-EGFR antibody. Precipitated proteins (top) and total cell lysates (bottom) were immunoblotted with anti-HA antibody. The membrane from the top blot was reprobed with anti-EGFR antibody to confirm equal levels of protein loading. FL, full-length. (C) COS cells were transiently transfected with the indicated myc–Abi-1 plasmids. Grb2 was immunoprecipitated from cell lysates. Precipitated proteins (top) and total cell lysates (bottom) were immunoblotted with anti-myc antibody. The membrane from the top blot was reprobed with anti-Grb2 antibody to confirm equal levels of protein loading (middle).
- Fig. 4.
Inhibition by Abi-1 of EGF-induced Erk2 activation but not EGF-induced JNK or Akt activation. (A) COS cells were transiently cotransfected with the indicated HA-Erk2 and myc–Abi-1 plasmids. Serum-starved cells were left untreated or were stimulated with EGF (100 ng/ml) for 5 min. HA-Erk2 was immunoprecipitated from cell lysates with anti-HA antibody (αHA) and immunoblotted with antibodies against active Erks (top). The membrane was reprobed with anti-HA antibody to confirm equal levels of protein loading (middle). Total cell lysates were probed with anti-myc antibody to confirm expression of myc-tagged Abi-1 proteins (bottom). FL, full length; IP, immunoprecipitate; WB, Western blot. (B) The experiment whose results are shown here was performed with the indicated plasmids as described for panel A except that EGF treatment was for 1, 2, 5, 10, or 20 min. The relative normalized amount of active Erk is shown beneath each lane. Graphic plots of relative amounts of active Erk versus time are included for both the upper and lower blots. (C) The experiment whose results are shown here was performed with the indicated plasmids as described for panel A except that EGF treatment was for 20, 40, or 60 min. The relative normalized amount of active Erk is shown beneath each lane. (D) The experiment whose results are shown here was performed as described for panel A except that HA-JNK and antibodies against active JNK replaced HA-Erk2 and antibodies against active Erks, respectively. (E) The experiment whose results are shown here was performed as described for panel A except that HA-Akt and antibodies against active Akt replaced HA-Erk2 and antibodies against active Erks, respectively.
- Fig. 5.
Inhibition of v-Abl-induced Erk2 activation by Abi-1. 293T cells were transiently cotransfected with plasmids encoding HA-Erk2, wild-type (WT) p160v-abl, kinase-dead (KD) p160v-abl, or v-Src, and full-length HA–Abi-1 (FL) or HA–AbiΔ1–85 (Δ1–85). Transfected cells were serum starved in 0.2% FBS for 24 h before lysis. Cell lysates were probed with antibodies against active Erks (αActive Erk) (top), HA (middle), and Abl (bottom). Expression of v-Src was not detected by our methods. WB, Western blot.
- Fig. 6.
Tyrosine phosphorylation of endogenous Abi-1 induced by v-Abl. (A) Serum-starved D5 cells were left at 39°C (nonpermissive temperature) or shifted to 32°C (permissive temperature) for 1 h. Abi-1 was immunoprecipitated from cell lysates and immunoblotted with antiphosphotyrosine antibody (αPtyr) (top). The membranes were reprobed with anti-Abi-1 antibodies to confirm equal levels of protein loading (bottom). The arrows indicate the position of Abi-1. N and P denote nonpermissive and permissive temperatures, respectively. WB, Western blot. (B) Growth factor-deprived parental BAF/3 cells and BAF/3 cells stably transfected with p160v-abl were left untreated or were stimulated with IL-3 (50 ng/ml). Abi-1 was immunoprecipitated and immunoblotted as described for panel A. IP, immunoprecipitate.
- Fig. 7.
Tyrosine phosphorylation of endogenous Abi-1 induced by serum stimulation. Serum-starved BALB/c3T3 cells were stimulated with 20% FBS for the indicated times (in minutes). Abi-1 was immunoprecipitated from cell lysates with anti-Abi-1 antibodies (αAbi) and immunoblotted with antiphosphotyrosine antibody (top). The membrane was reprobed with anti-Abi-1 antibodies to confirm equal levels of protein loading (bottom). The arrows indicate the position of Abi-1. PI, preimmune serum control; IP, immunoprecipitate; WB, Western blot.
Tables
- Table 1.
Yeast two-hybrid tests of the binding of the Abi-1 SH3 domain to the carboxy-terminal domain of Sos2
Protein moiety fused to LexADBDa Protein moiety fused to Gal4ADa β-Galactosidase activityb Abi-1(394–475) None − None Abl(4–1087) − Abi-1(394–475) Abl(4–1087) + Abi-1(394–475) Sos2(874–1297) + Abi-1(394–475) Sos2(974–1297) + None Sos2(874–1297) − None Sos2(974–1297) −
