Identification and characterization of a novel mammalian isoform of the endocytic adaptor ITSN1

Dergai, Mykola; Skrypkina, Inessa; Dergai, Oleksandr; Tsyba, L. O.; Novokhatska, Olga; Filonenko, Valeriy; Drobot, L. B.; Rynditch, A. V.

doi:10.1016/j.gene.2011.06.021

Cited by 13 publications

(18 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Immunostaining of the cells was performed as described previously [31]. For immunofluorescence, Texas Red-conjugated goat anti-rabbit (Vector Laboratories Inc.), Alexa 488-conjugated goat anti-rabbit or Alexa 488-conjugated donkey anti-mouse secondary antibodies (Molecular Probes) were used.…”

Section: Methodsmentioning

confidence: 99%

Adaptor Proteins Intersectin 1 and 2 Bind Similar Proline-Rich Ligands but Are Differentially Recognized by SH2 Domain-Containing Proteins

et al. 2013

Self Cite

View full text Add to dashboard Cite

BackgroundScaffolding 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.Methodology/Principal FindingsWe 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/SignificanceOur 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.

show abstract

Section: Methodsmentioning

confidence: 99%

Adaptor Proteins Intersectin 1 and 2 Bind Similar Proline-Rich Ligands but Are Differentially Recognized by SH2 Domain-Containing Proteins

et al. 2013

Self Cite

View full text Add to dashboard Cite

show abstract

“…Ano- ther possibility is bringing together signaling regulators SPRY2 and Cbl that are functionally linked and both interact with the SH3 domains of ITSN1. Our previous results showed that ITSN1 and Cbl form a complex in vivo independently of growth factor stimulation [5]. Contrary to this ITSN1 and SPRY2 displayed distinct localization in quiescent cells but partially overlapped when supplemented with FBS.…”

Section: Materials and Methods Expression Constructs And Antibodiesmentioning

confidence: 81%

“…Cell culture and confocal microscopy. Adherent cell lines HEK293 and MCF-7 cells were maintained and transfected as described [5]. For immunofluorescent analysis fixed MCF-7 cells were incubated with the appropriate antibodies.…”

Section: Materials and Methods Expression Constructs And Antibodiesmentioning

confidence: 99%

RTK signaling regulator SPRY2 associates with endocytic adaptor ITSN1 in vivo

et al. 2012

View full text Add to dashboard Cite

Mitogenic signaling from RTK (receptor tyrosine kinase) is tightly linked to endocytosis that regulates the fidelity of signal transduction. Intersectin 1 (ITSN1) is an evolutionarily conserved adaptor implicated in the earliest stages of clathrin-mediated endocytosis and involved in malignant transformation when overexpressed. The aim of this study was to find proteins that could connect ITSN1 to RTK signaling. Methods. In silico prediction of interaction motifs, expression of proteins in bacterial system and mammalian cell culture, immunoprecipitation, GST pull-down analysis, immunofluorescent studies. Results. Using «Scansite» service a binding of ITSN1 SH3A domain to signaling protein SPRY2 was predicted. For experimental verification of this interaction the GST fu- sion SH3 domains of ITSN1 were produced and purified from Escherichia coli. GST pull-down analysis showed that SH3A, N-SH3A (neuron-specific variant), SH3C and SH3E domains of ITSN1 were able to pull-down Flag- SPRY2 from cell lysate. Immunofluorescent analysis together with coimmunoprecipitation indicated that Omni- ITSN1 and Flag-SPRY2 were partially colocalized and formed a complex in vivo. Conclusions. Our data showed that endocytic adaptor ITSN1 and signaling protein SPRY2 are associated in vivo presumably via PRD/SH3 interaction

show abstract

“…This finding may explain relatively low abundance of ITSN1‐22a when compared to ITSN1‐s. Previously , we suggested that ITSN1‐22a may serve as a dominant‐negative variant of ITSN1‐s, and under certain conditions degradation could be paused and ITSN1‐22a may play an inhibitory role. Low levels of ITSN1‐22a were observed in epithelial (Hela, MCF7) and fibroblast (HEK293) cell lines, in muscle and a liver among mouse tissues .…”

Section: Discussionmentioning

confidence: 98%

Ubiquitin‐ligase AIP4 controls differential ubiquitination and stability of isoforms of the scaffold protein ITSN1

Dergai

Rynditch

2018

FEBS Letters

Self Cite

View full text Add to dashboard Cite

At present, the role of ubiquitination of cargoes internalized from the plasma membrane is better understood than the consequences of ubiquitination of proteins comprising the endocytic machinery. Here, we show that the E3 ubiquitin ligase AIP4/ITCH contributes to the differential ubiquitination of isoforms of the endocytic scaffold protein intersectin1 (ITSN1). The major isoform ITSN1-s is monoubiquitinated, whereas the minor one, ITSN1-22a undergoes a combination of mono- and oligoubiquitination. The monoubiquitination is required for ITSN1-s stability, whereas the oligoubiquitination of ITSN1-22a causes its proteasomal degradation. This explains the observed low abundance of the minor isoform in cells. Thus, different modes of ubiquitination regulated by AIP4 have opposite effects on ITSN1 isoform stability.

show abstract

Identification and characterization of a novel mammalian isoform of the endocytic adaptor ITSN1

Cited by 13 publications

References 23 publications

Adaptor Proteins Intersectin 1 and 2 Bind Similar Proline-Rich Ligands but Are Differentially Recognized by SH2 Domain-Containing Proteins

Adaptor Proteins Intersectin 1 and 2 Bind Similar Proline-Rich Ligands but Are Differentially Recognized by SH2 Domain-Containing Proteins

RTK signaling regulator SPRY2 associates with endocytic adaptor ITSN1 in vivo

Ubiquitin‐ligase AIP4 controls differential ubiquitination and stability of isoforms of the scaffold protein ITSN1

Contact Info

Product

Resources

About