Comprehensive analysis of interactions between the Src-associated protein in mitosis of 68 kDa and the human Src-homology 3 proteome

PLoS One. 2012;7(6):e38540. doi: 10.1371/journal.pone.0038540. Epub 2012 Jun 20.


The protein Sam68 is involved in many cellular processes such as cell-cycle regulation, RNA metabolism, or signal transduction. Sam68 comprises a central RNA-binding domain flanked by unstructured tails containing docking sites for signalling proteins including seven proline-rich sequences (denoted P0 to P6) as potential SH3-domain binding motifs. To comprehensively assess Sam68-SH3-interactions, we applied a phage-display screening of a library containing all approx. 300 human SH3 domains. Thereby we identified five new (from intersectin 2, the osteoclast stimulating factor OSF, nephrocystin, sorting nexin 9, and CIN85) and seven already known high-confidence Sam68-ligands (mainly from the Src-kinase family), as well as several lower-affinity binders. Interaction of the high-affinity Sam68-binders was confirmed in independent assays in vitro (phage-ELISA, GST-pull-down) and in vivo (FACS-based FRET-analysis with CFP- and YFP-tagged proteins). Fine-mapping analyses with peptides established P0, P3, P4, and P5 as exclusive docking-sites for SH3 domains, which showed varying preferences for these motifs. Mutational analyses identified individual residues within the proline-rich motifs being crucial for the interactions. Based on these data, we generated a Sam68-mutant incapable of interacting with SH3 domains any more, as subsequently demonstrated by FRET-analyses. In conclusion, we present a thorough characterization of Sam68's interplay with the SH3 proteome. The observed interaction between Sam68 and OSF complements the known Sam68-Src and OSF-Src interactions. Thus, we propose, that Sam68 functions as a classical scaffold protein in this context, assembling components of an osteoclast-specific signalling pathway.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism*
  • Adaptor Proteins, Vesicular Transport / genetics
  • Adaptor Proteins, Vesicular Transport / metabolism
  • Blotting, Western
  • Cell Line
  • Cytoskeletal Proteins
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Enzyme-Linked Immunosorbent Assay
  • Humans
  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Peptides / genetics
  • Peptides / metabolism
  • Protein Binding
  • Proteome / genetics
  • Proteome / metabolism*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Signal Transduction
  • Sorting Nexins / genetics
  • Sorting Nexins / metabolism
  • src Homology Domains / genetics
  • src Homology Domains / physiology*


  • Adaptor Proteins, Signal Transducing
  • Adaptor Proteins, Vesicular Transport
  • Cytoskeletal Proteins
  • DNA-Binding Proteins
  • ITSN2 protein, human
  • Intracellular Signaling Peptides and Proteins
  • KHDRBS1 protein, human
  • Membrane Proteins
  • NPHP1 protein, human
  • Peptides
  • Proteome
  • RNA-Binding Proteins
  • SH3KBP1 protein, human
  • SNX9 protein, human
  • Sorting Nexins
  • osteoclast stimulating factor