The integrity of the SH3 binding motif of AFAP-110 is required to facilitate tyrosine phosphorylation by, and stable complex formation with, Src.
The actin filament-associated protein AFAP-110 forms a stable complex with activated variants of Src in chick embryo fibroblast cells. Stable complex formation requires the integrity of the Src SH2 and SH3 domains. In addition, AFAP-110 encodes two adjacent SH3 binding motifs and six candidate SH2 binding motifs. These data indicate that both SH2 and SH3 domains may work cooperatively to facilitate Src/AFAP-110 stable complex formation. As a test for this hypothesis, we sought to understand whether one or both SH3 binding motifs in AFAP-110 modulate interactions with the Src SH3 domain and if this interaction was required to present AFAP-110 for tyrosine phosphorylation by, and stable complex formation with, Src. A proline to alanine site-directed mutation in the amino terminal SH3 binding motif (SH3bm I) was sufficient to abrogate absorption of AFAP-110 with GST-SH3STC. Co-expression of activated Src (pp60(527F)) with AFAP-110 in Cos-1 cells permit tyrosine phosphorylation of AFAP-110 and stable complex formation with pp60(527F). However, co-expression of the SH3 null-binding mutant (AFAP71A) with pp60(527F) revealed a 2.7 fold decrease in steady-state levels of tyrosine phosphorylation, compared to AFAP-110. Although a lower but detectable level of AFAP71A was phosphorylated on tyrosine, AFAP71A could not be detected in stable complex with pp60(527F), unlike AFAP-110. These data indicate that SH3 interactions facilitate presentation of AFAP-110 for tyrosine phosphorylation and are also required for stable complex formation with pp60(527F).
Guappone
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Mary Babb Randolph Cancer Center, Morgantown, WV, USA.
Flynn
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DC
D019943:Amino Acid Substitution; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D003001:Cloning, Molecular; D005982:Glutathione Transferase / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism*; D016296:Mutagenesis; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains / Q000235:genetics / Q000502:physiology*
C084510:AFAP 110; D008840:Microfilament Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D005982:Glutathione Transferase