Adaptor proteins intersectin 1 and 2 bind similar proline-rich ligands but are differentially recognized by SH2 domain-containing proteins.
BACKGROUND
Scaffolding proteins of the intersectin (ITSN) family, ITSN1 and ITSN2, are crucial for the initiation stage of clathrin-mediated endocytosis. These proteins are closely related but have implications in distinct pathologies. To determine how these proteins could be separated in certain cell pathways we performed a comparative study of ITSNs.


RESULTS
We have shown that endogenous ITSN1 and ITSN2 colocalize and form a complex in cells. A structural comparison of five SH3 domains, which mediated most ITSNs protein-protein interactions, demonstrated a similarity of their ligand-binding sites. We showed that the SH3 domains of ITSN2 bound well-established interactors of ITSN1 as well as newly identified ITSNs protein partners. A search for a novel interacting interface revealed multiple tyrosines that could be phosphorylated in ITSN2. Phosphorylation of ITSN2 isoforms but not ITSN1 short isoform was observed in various cell lines. EGF stimulation of HeLa cells enhanced tyrosine phosphorylation of ITSN2 isoforms and enabled their recognition by the SH2 domains of the Fyn, Fgr and Abl1 kinases, the regulatory subunit of PI3K, the adaptor proteins Grb2 and Crk, and phospholipase C gamma. The SH2 domains mentioned were unable to bind ITSN1 short isoform.


CONCLUSIONS
Our results indicate that during evolution of vertebrates ITSN2 acquired a novel protein-interaction interface that allows its specific recognition by the SH2 domains of signaling proteins. We propose that these data could be important to understand the functional diversity of paralogous ITSN proteins.
Department of Functional Genomics, Institute of Molecular Biology and Genetics, Kyiv, Ukraine.
Olga
O
Novokhatska
Mykola
M
Dergai
Liudmyla
L
Tsyba
Inessa
I
Skrypkina
Valeriy
V
Filonenko
Jacques
J
Moreau
Alla
A
Rynditch
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D002966:Clathrin / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000235:genetics; D004815:Epidermal Growth Factor / Q000494:pharmacology; D019143:Evolution, Molecular; D005786:Gene Expression Regulation; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011392:Proline / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; C485568:ITSN1 protein, human; C509776:ITSN2 protein, human; D008024:Ligands; D004815:Epidermal Growth Factor; D011392:Proline