RTK signaling regulator SPRY 2 associates with endocytic adaptor ITSN 1 in vivo

Aim. 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 fusion 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 FlagSPRY2 from cell lysate. Immunofluorescent analysis together with coimmunoprecipitation indicated that OmniITSN1 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.

Introduction.Intersectin 1 (ITSN1) is conserved from nematodes to humans multidomain adaptor that is crucial for the initiation of clathrin-mediated endocytosis.Depletion of highly similar ITSN1 and ITSN2 scaffolding molecules together with Eps15 proteins abolishes clathrin-coated pits assembly [1].Clathrin-mediated endocytosis is a common mechanism for internalization of activated receptor tyrosine kinases (RTKs) and regulation of mitogenic signaling.
Endocytic adaptor ITSN1 is involved in intracellular signal transduction through interaction with Ras activator SOS1, ubiquitin ligase Cbl and signaling protein CIN85 [2].It was reported that overexpression of ITSN1 results in fibroblasts malignant transformation [3].The aim of this study was to find other signaling molecules that could link ITSN1 to mitogenic signaling.
Binding assays and Western blot analysis.The GST fusion proteins were purified on glutathione-Sepharose 4B beads («GE Healthcare»).Lysates of HEK293 cells were prepared in extraction buffer that contained 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 % Triton X-100, 1 mM PMSF.For pull-down assays, immobilized on beads GST-SH3 domains were incubated with cell lysates for 1 h at 4 °C.For immunoprecipitation (IP), the cell lysate was incubated with antibodies and protein A/G Agarose beads («Santa Cruz Biotechnology») for 2 h.Bound proteins were subjected to SDS-PAGE and immunoblotting.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.Confocal images were taken using a Zeiss LSM510 microscope.
Results and discussion.Analysis of available data in the field of signaling from activated RTKs suggested SPRY2 (Sprouty 2) as a possible candidate for ITSN1 binding.This protein belongs to a family of SPRY proteins known to regulate signal transduction from flies to mammals [6].SPRY2 possesses cysteine-rich domain common for all family members and contains at the Cterminus several proline residues (PRD) that could represent interaction motif for the SH3 domains.Using «Scansite» service (www.scansite.mit.edu) a binding of the SH3A domain to proline in position 307 of SPRY2 molecule was predicted.To test this possibility, the affinity purified GST-fused SH3 domains of ITSN1 were used to pull down Flag-SPRY2 from lysate of HEK293 cells expressing this protein.Obtained results showed that the SH3A, as well as its neuron-specific variant (N-SH3A), SH3C and SH3E domains interacted with Flagtagged SPRY2 in vitro (Fig. 1, A).To study this interaction in vivo HEK293 cells were cotransfected with constructs encoding Omni-ITSN1 and Flag-SPRY2.Using antibodies against Omni-tag SPRY2 was precipitated from cell lysate together with ITSN1 (Fig. 1, B).
Immunofluorescent studies were used to investigate the intracellular distribution of ITSN1/SPRY2 protein complexes.In quiescent cells SPRY2 is associated with microtubules and translocates to the membrane ruffles upon EGF-stimulation [7].In serum-starved cells coexpressing Omni-ITSN1 and Flag-SPRY2 these proteins were spatially separated (Fig. 2, A) but displayed partial colocalization when supplemented with FBS (Fig. 2, B).
Accumulated data indicate that SPRY proteins are sensors and integrators of signaling [6].In this study we demonstrated an in vivo complex formation between SPRY2 and endocytic adaptor ITSN1.A direct interaction between these molecules was mediated by binding of proline-rich motif of SPRY2 to the SH3 domains of ITSN1.Regarding that several SH3 domains were able to pull down signaling protein, a role of ITSN1 in clustering a few SPRY2 molecules could be suggested.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.Obtained data could suggest recruitment of SPRY2 to preexisting ITSN1/Cbl complex after growth factor stimulation.This question remains a challenge for future investigations.

Ó
Institute of Molecular Biology and Genetics, NAS of Ukraine, 2012