Intersectin goes nuclear: secret life of an endocytic protein

Abstract

Intersectin 1-short (ITSN1-s) is a 1220 amino acid ubiquitously expressed scaffold protein presenting a multidomain structure that allows to spatiotemporally regulate the functional interaction of a plethora of proteins. Besides its well-established role in endocytosis, ITSN1-s is involved in the regulation of cell signaling and is implicated in tumorigenesis processes, although the signaling pathways involved are still poorly understood. Here, we identify ITSN1-s as a nucleocytoplasmic trafficking protein. We show that, by binding to importin (IMP)α, a small fraction of ITSN1-s localizes in the cell nucleus at the steady state, where it preferentially associates with the nuclear envelope and interacts with lamin A/C. However, upon pharmacological ablation of chromosome region maintenance 1 (CRM-1)-dependent nuclear export pathway, the protein accumulates into the nucleus, thus revealing its moonlighting nature. Analysis of deletion mutants revealed that the coiled coil (CC) and Src homology (SH3) regions play the major role in its nucleocytoplasmic shuttling. While no evidence of nuclear localization signal (NLS) was detected in the CC region, a functional bipartite NLS was identified within the SH3D region of ITSN1-s (RKKNPGGWWEGELQARGKKRQIGW-1127), capable of conferring energy-dependent nuclear accumulation to reporter proteins and whose mutational ablation affects nuclear import of the whole SH3 region. Thus, ITSN1-s is an endocytic protein, which shuttles between the nucleus and the cytoplasm in a CRM-1- and IMPα-dependent fashion.

Scaffold proteins ITSN1 and ITSN2 interact with nuclear RNA-binding proteins

“…Previously, the analysis of HeLa nuclei phosphoproteome identified ITSN1 peptides in the nuclei of HeLa cells [14]. More recently, ITSN1 has been found to undergo nucleus-cytoplasmic shuttling via CRM1-and importin α-dependent pathways and has been shown to co-localize with lamin A/C [15]. The data supports our observations revealing nuclear localization of ITSN1 (unpublished data).…”

ITSN1 regulates SAM68 solubility through SH3 domain interactions with SAM68 proline-rich motifs

“…Consistent with this view, ITSN1 has been associated with neuronal diseases [31,32]. However, ITSN1 has been also found in the cell nucleus with unknown functions [33]. The identification of several RBPs as potential ITSN1 partners by previous high throughput screenings [34][35][36] led us to hypothesize that the SH3 domains of ITSN1 could be involved in RBP processing in the nucleus.…”

“…ITSN1 is a cytoplasmic scaffold protein that has also been identified in the cell nucleus [33]. To further clarify the nuclear location of ITSN1, we visualized the spatial distribution of endogenous ITSN1 in HeLa cells using specific anti-ITSN1 antibodies.…”

Generation of Combinatorial Lentiviral Vectors Expressing Multiple Anti-Hepatitis C Virus shRNAs and Their Validation on a Novel HCV Replicon Double Reporter Cell Line

“…Plasmid RLuc-UL44, mediating expression of a N-terminal fusion of Renilla Luciferase (RLuc) to human cytomegalovirus DNA polymerase processivity factor UL44 [ 34 ], was used as template for amplification with primers FW-RLuc (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTGACCAGCAAGGTGTACGAC-3′) and RV-RLuc (5′-GGGGACCACTTTGTACAAGAAAGCTGGGTTTAGAGATCCCCCTGCTCGTTC-3′) to generate a PCR product containing the RLuc coding sequence flanked by Gateway compatible attB sites. The purified PCR product was used with plasmid pDNR207 (Thermofisher Scientific, Waltham, MA, USA) for a Gateway BP recombination reaction to generate entry clone pDNR-RLuc, as described in [ 35 ]. Entry clone pDNR-RLuc was used with the lentiviral Expression vector pWPI_nHA_puro_rfB for Gateway LR recombination reactions as described previously [ 36 ], to generate Lentiviral expression vector pWPI_nHA_RLuc_puro, mediating expression of N-terminally HA-tagged RLuc under the control of the EF1α promoter [ 37 ], Packaging plasmid pCMVΔ8.74 (Addgene #22036) and the VSV envelope glycoprotein expression vector pMD2.G (Addgene #12259) were kindly provided by Didier Trono (Lausanne, Switzerland).…”