The atypical Rho GTPase RhoU interacts with intersectin-2 to regulate endosomal recycling pathways.

Rho GTPases play a key role in various membrane trafficking processes. RhoU is an atypical small Rho GTPase related to Rac/Cdc42, which possesses unique N- and C-terminal domains that regulate its function and its subcellular localization. RhoU localizes at the plasma membrane, on endosomes and in cell adhesion structures where it governs cell signaling, differentiation and migration. However, despite its endomembrane localization, RhoU function in vesicular trafficking has been unexplored. Here, we identified intersectins (ITSNs) as new binding partners for RhoU and showed that the second PxxP motif at the N terminus of RhoU mediated interactions with the SH3 domains of ITSNs. To evaluate the function of RhoU and ITSNs in vesicular trafficking, we used fluorescent transferrin as a cargo for uptake experiments. We showed that silencing of either RhoU or ITSN2, but not ITSN1, increased transferrin accumulation in early endosomes, resulting from a defect in fast vesicle recycling. Concomitantly, RhoU and ITSN2 colocalized to a subset of Rab4-positive vesicles, suggesting that a RhoU-ITSN2 interaction may occur on fast recycling endosomes to regulate the fate of vesicular cargos.

WIP/ITSN1 complex is involved in cellular vesicle trafficking and formation of filopodia-like protrusions.

WIP (WASP interacting protein) together with N-WASP (neural Wiskott-Aldrich syndrome protein) regulates actin polymerization that is crucial for invadopodia and filopodia formation. Recently, we reported the WIP interaction with ITSN1 which is highly implicated in endo-/exocytosis, apoptosis, mitogenic signaling and cytoskeleton rearrangements. Here we demonstrate that the WIP/ITSN1 complex is involved in the transferrin receptor recycling and partially co-localizes with a marker of the fast recycling endosomes, RAB4. Moreover, ITSN1 recruits WIP to RAB4-positive vesicles upon overexpression. Our data indicate that WIP enhances the interaction of N-WASP with ITSN1 and promotes ITSN1/ß-actin association. Moreover, the WIP/ITSN1-L complex facilitates formation of filopodia-like protrusions in MCF-7 cells. Thus, WIP/ITSN1 complex is involved in the cellular vesicle trafficking and actin-dependent membrane processes.

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

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.

Mammalian verprolin CR16 acts as a modulator of ITSN scaffold proteins association with actin.

Actin cytoskeleton rearrangements are required for normal cell functioning, and their deregulation leads to various pathologies. Members of two mammalian protein families - ITSNs (ITSN1 and ITSN2) and verprolins (WIP, CR16 and WIRE) are involved in Cdc42/N-WASP/Arp2/3 signaling pathway-mediated remodeling of the actin cytoskeleton. Recently we demonstrated that ITSNs interact with the actin-regulating protein WIP. Here, we show that other member of verprolin family, CR16, also forms complexes with ITSN1 and ITSN2 in human cell lines. The actin-binding protein CR16 modulates ITSN/ß-actin association. Moreover, overexpressed CR16 promoted co-localization of ITSN1 with F-actin in MCF-7 breast cancer cells. Our data demonstrated that CR16 mRNA is expressed in glioblastoma and breast tumors. These findings provide the basis for further functional investigations of the ITSN/CR16 complex that may play an important role in actin remodeling and cellular invasion.

Concentration and Methylation of Cell-Free DNA from Blood Plasma as Diagnostic Markers of Renal Cancer.

The critical point for successful treatment of cancer is diagnosis at early stages of tumor development. Cancer cell-specific methylated DNA has been found in the blood of cancer patients, indicating that cell-free DNA (cfDNA) circulating in the blood is a convenient tumor-associated DNA marker. Therefore methylated cfDNA can be used as a minimally invasive diagnostic marker. We analysed the concentration of plasma cfDNA and methylation of six tumor suppressor genes in samples of 27 patients with renal cancer and 15 healthy donors as controls. The cfDNA concentrations in samples from cancer patients and healthy donors was measured using two different methods, the SYBR Green I fluorescence test and quantitative real-time PCR. Both methods revealed a statistically significant increase of cfDNA concentrations in cancer patients. Hypermethylation on cfDNA was detected for the LRRC3B (74.1%), APC (51.9%), FHIT (55.6%), and RASSF1 (62.9%) genes in patients with renal cancer. Promoter methylation of VHL and ITGA9 genes was not found on cfDNA. Our results confirmed that the cfDNA level and methylation of CpG islands of RASSF1A, FHIT, and APC genes in blood plasma can be used as noninvasive diagnostic markers of cancer.

Evolutionary Changes on the Way to Clathrin-Mediated Endocytosis in Animals.

Endocytic pathways constitute an evolutionarily ancient system that significantly contributed to the eukaryotic cell architecture and to the diversity of cell type-specific functions and signaling cascades, in particular of metazoans. Here we used comparative proteomic studies to analyze the universal internalization route in eukaryotes, clathrin-mediated endocytosis (CME), to address the issues of how this system evolved and what are its specific features. Among 35 proteins crucially required for animal CME, we identified a subset of 22 proteins common to major eukaryotic branches and 13 gradually acquired during evolution. Based on exploration of structure-function relationship between conserved homologs in sister, distantly related and early diverged branches, we identified novel features acquired during evolution of endocytic proteins on the way to animals: Elaborated way of cargo recruitment by multiple sorting proteins, structural changes in the core endocytic complex AP2, the emergence of the Fer/Cip4 homology domain-only protein/epidermal growth factor receptor substrate 15/intersectin functional complex as an additional interaction hub and activator of AP2, as well as changes in late endocytic stages due to recruitment of dynamin/sorting nexin 9 complex and involvement of the actin polymerization machinery. The evolutionary reconstruction showed the basis of the CME process and its subsequent step-by-step development. Documented changes imply more precise regulation of the pathway, as well as CME specialization for the uptake of specific cargoes and cell type-specific functions.

Intersectin adaptor proteins are associated with actin-regulating protein WIP in invadopodia.

Invasive cancer cells form actin-rich membrane protrusions called invadopodia that degrade extracellular matrix and facilitate cell invasion and metastasis. WIP (WASP-interacting protein) together with N-WASP (neural Wiskott-Aldrich syndrome protein) are localized in invadopodia and play a crucial role in their formation. Here we show that WIP interacts with endocytic adaptor proteins of the intersectin (ITSN) family, ITSN1 and ITSN2. The interaction is mediated by the SH3 domains of ITSNs and the middle part of the WIP proline-rich motifs. We have also demonstrated that ITSN1, WIP and N-WASP can form a complex in cells. Endogenous ITSN1 and ITSN2 are located in invasive protrusions of MDA-MB-231 breast cancer cell line. Moreover, data from immunofluorescent analysis revealed co-localization of ITSN1 and WIP at sites of invadopodia formation and in clathrin-coated pits. Together, these findings provide insights into the molecular mechanisms of invadopodia formation and identify ITSNs as scaffold proteins involved in this process.

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. METHODOLOGY/PRINCIPAL FINDINGS: 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/SIGNIFICANCE: 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.

Intersectin: The Crossroad between Vesicle Exocytosis and Endocytosis.

Intersectins (ITSNs) are a family of highly conserved proteins with orthologs from nematodes to mammals. In vertebrates, ITSNs are encoded by two genes (itsn1 and itsn2), which act as scaffolds that were initially discovered as proteins involved in endocytosis. Further investigation demonstrated that ITSN1 is also implicated in several other processes including regulated exocytosis, thereby suggesting a role for ITSN1 in the coupling between exocytosis and endocytosis in excitatory cells. Despite a high degree of conservation amongst orthologs, ITSN function is not so well preserved as they have acquired new properties during evolution. In this review, we will discuss the role of ITSN1 and its orthologs in exo- and endocytosis, in particular in neurons and neuroendocrine cells.

The LMP2A protein of Epstein-Barr virus regulates phosphorylation of ITSN1 and Shb adaptors by tyrosine kinases.

Latent Membrane Protein 2A (LMP2A) is an Epstein-Barr virus-encoded protein that is important for the maintenance of latent infection. Its activity affects cellular differentiation, migration, proliferation and B cell survival. LMP2A resembles a constitutively activated B cell antigen receptor and exploits host kinases to activate a set of downstream signaling pathways. In the current study we demonstrate the interaction of LMP2A with intersectin 1 (ITSN1), a key endocytic adaptor protein. This interaction occurs via both the N- and C-tails of LMP2A and is mediated by the SH3 domains of ITSN1. Additionally, we identified the Shb adaptor and the Syk kinase as novel binding ligands of ITSN1. The Shb adaptor interacts simultaneously with the phosphorylated tyrosines of LMP2A and the SH3 domains of ITSN1 and mediates indirect interaction of ITSN1 to LMP2A. Syk kinase promotes phosphorylation of both ITSN1 and Shb adaptors in LMP2A-expressing cells. In contrast to ITSN1, Shb phosphorylation depends additionally on Lyn kinase activity. Considering that Shb and ITSN1 are implicated in various receptor tyrosine kinase signaling, our results indicate that LMP2A can affect a number of signaling pathways by regulating the phosphorylation of the ITSN1 and Shb adaptors.

Alterations of the WNT7A gene in clear cell renal cell carcinomas.

WNT7A (wingless-type MMTV integration site family, member 7A) is a known tumor suppressor gene of non-small cell lung carcinomas (NSCLC) and is frequently inactivated due to CpG-island hypermethylation in human cancers. The members of WNT family are involved in cell signaling and play crucial roles in cancer development. In the present work hypermethylation of the WNT7A gene was detected in 66% (29/44) of analyzed clear cell renal cell carcinomas (RCCs) using methyl-specific PCR (MSP). Moreover, bisulfite sequencing confirmed intensive hypermethylation of the 5'-CpG island of the WNT7A gene. Methylation analysis revealed positive correlations between tumor stage, Fuhrman nuclear grade and WNT7A hypermethylation. Additionally, restoration of WNT7A gene expression in the A498 cell line by 5-aza-2'-deoxycytidine treatment confirmed a direct contribution of hypermethylation in silencing of the WNT7A gene. High frequency of loss of heterozygosity (LOH) was demonstrated on chromosome 3p25 in regions surrounding the WNT7A gene. The frequent down-regulation of WNT7A gene expression was detected in 88% (15/17) of clear cell RCCs. We have also shown that the WNT7A gene possesses tumor suppression function by colony-formation and cell proliferation assays in RCC cell lines. In summary, the WNT7A gene is inactivated by genetic/epigenetic alterations in clear cell RCC and demonstrates tumor suppressor properties.

Endocytic adaptor protein intersectin 1 forms a complex with microtubule stabilizer STOP in neurons.

Intersectin 1 (ITSN1) is a multidomain adaptor protein that functions in clathrin-mediated endocytosis and signal transduction. This protein is highly abundant in neurons and is implicated in Down syndrome, Alzheimer's disease and, possibly, other neurodegenerative disorders. Here we used an in vitro binding assay combined with MALDI-TOF mass spectrometry to identify novel binding partners of ITSN1. We found that the neuron-specific isoform of the stable tubule-only polypeptide (STOP) interacts with SH3A domain of ITSN1. STOP and ITSN1 were shown to form a complex in vivo and to partially co-localize in rat primary hippocampal neurons. As STOP is a microtubule-stabilizing protein that is required for several forms of synaptic plasticity in the hippocampus, identification of this interaction raises the possibility of ITSN1 participation in this process.

LRRC3B gene is frequently epigenetically inactivated in several epithelial malignancies and inhibits cell growth and replication.

Chromosome 3 specific NotI microarrays containing 180 NotI linking clones associated with 188 genes were hybridized to NotI representation probes prepared using matched tumor/normal samples from major epithelial cancers: breast (47 pairs), lung (40 pairs) cervical (43 pairs), kidney (34 pairs of clear cell renal cell carcinoma), colon (24 pairs), ovarian (25 pairs) and prostate (18 pairs). In all tested primary tumors (compared to normal controls) methylation and/or deletions was found. For the first time we showed that the gene LRRC3B was frequently methylated and/or deleted in breast carcinoma - 32% of samples, cervical - 35%, lung - 40%, renal - 35%, ovarian - 28%, colon - 33% and prostate cancer - 44%. To check these results bisulfite sequencing using cloned PCR products with representative two breast, one cervical, two renal, two ovarian and two colon cancer samples was performed. In all cases methylation was confirmed. Expression analysis using RT-qPCR showed that LRRC3B is strongly down-regulated at the latest stages of RCC and ovarian cancers. In addition we showed that LRRC3B exhibit strong cell growth inhibiting activity (more than 95%) in colony formation experiments in vitro in KRC/Y renal cell carcinoma line. All these data suggest that LRRC3B gene could be involved in the process of carcinogenesis as a tumor suppressor gene.

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

Intersectin 1 (ITSN1) is an evolutionarily conserved adaptor protein engaged in clathrin-mediated endocytosis, cell signaling and actin cytoskeleton rearrangements. Two major ITSN1 isoforms were initially described, the ubiquitous short isoform (ITSN1-s) and the long isoform (ITSN1-l) expressed predominantly in neurons. Numerous alternative splicing events for ITSN1 pre-mRNA were later identified. Here we describe a novel isoform ITSN1-22a with an alternative C-terminus encoded by exon 22a. This exon is only found in placental mammals. The transcript of ITSN1-22a is detected in a wide range of human and mouse tissues. We show here that two alternative splicing events affect the coding sequence of the ITSN1-22a isoform. Moreover, alternative polyadenylation of these transcripts was demonstrated in human tissues. The protein encoded by the ITSN1-22a transcript possesses two EH domains, a coiled-coil region, an SH3A domain and a specific C-terminal domain (CTD) but lacks four SH3 domains in comparison with ITSN1-s. The level of ITSN1-22a protein varies in different mouse tissues and human cell lines. The highest amounts of this isoform occur in mouse brain, spleen, lung and the human B cell line DG75. ITSN1-22a binds via its CTD to the SH3 domain of the endocytic protein amphiphysin 1 and the SH3A domain of ITSN1. Furthermore association in vivo and codistribution of ITSN1-22a and ITSN1-s were demonstrated suggesting that these isoforms could function in concert. We have revealed differential binding of ITSN1-s and ITSN1-22a to the ubiquitin ligase Cbl. Both isoforms possess the SH3A domain capable of binding to Cbl; however ITSN1-22a in contrast to ITSN1-s did not interact with Cbl in vivo. In vitro binding experiments demonstrated that the CTD of ITSN1-22a negatively regulated its binding to Cbl; at the same time interaction with another partner, dynamin 1 was not affected by the presence of the CTD. These data suggest that intramolecular interaction within ITSN1-22a could specifically regulate its binding to protein partners. Thus, this novel mammalian ITSN1 isoform possesses a significantly altered domain structure and performs specific protein-protein interactions.

Intersectin 2 nucleotide exchange factor regulates Cdc42 activity during Xenopus early development.

Intersectin 2 (ITSN2) is an evolutionarily conserved scaffold protein involved in endocytic internalization, regulation of actin cytoskeleton and epithelial morphogenesis. Recent studies of different Itsn-deficient organisms revealed that this gene is essential for the functioning of the nervous system and for organism viability. Here we report investigations on a possible role of the ITSN2 long isoform in the early embryonic development of Xenopus laevis. In vertebrates, alternative splicing generates several alternatively spliced isoforms of ITSN2. To date the long splice variant of ITSN2 (ITSN2-L) has been reported only for mammals. We show that transcripts of ITSN2-L can be detected in Xenopus embryos from the first cleavage onwards. Overexpression of functional domains of ITSN2-L in embryos resulted in aberrant phenotypes. The strongest phenotype was produced by the C-terminal extension of ITSN2-L. Embryos displayed hyperpigmentation and gastrulation failure that were incompatible with survival. The C-terminus of ITSN2-L includes the DH-PH tandem, a nucleotide exchange factor for the small GTPase Cdc42 and the C2 domain. Further investigations revealed that the DH-PH tandem was responsible for the development of the phenotype affecting the actin cytoskeleton in embryos. Observed developmental defects depended on Cdc42. The effect of expression of the constitutively active GTPase strongly resembled that of the DH-PH tandem. The dominant negative Cdc42 partially rescued developmental defects induced by the expression of the DH-PH tandem. Thus, our data indicate that the ITSN2 exchange factor regulates the activity of Cdc42 during embryo development affecting actin cytoskeleton in Xenopus embryos.

Intersectin multidomain adaptor proteins: regulation of functional diversity.

Adaptor/scaffold proteins serve as platforms for the assembly of multiprotein complexes and regulate the efficiency and specificity of signalling cascades. Intersectins (ITSNs) are an evolutionarily conserved adaptor protein family engaged in endo- and exocytosis, actin cytoskeleton rearrangement and signal transduction. This review summarizes recent advances in the function of ITSNs in neuronal and non-neuronal cells, the role of alternative splicing and alternative transcription in regulating the structural and functional diversity of ITSNs, their expression patterns in different tissues and during development, their interactions with proteins, as well as the potential relevance of ITSNs for neurodegenerative diseases and cancer. The diversity of mechanisms in the regulation of ITSN expression and specificity in different cells emphasizes the important role of ITSN proteins in vesicle trafficking and signalling.

Cloning and Initial Functional Characterization of Mlk4α and Mlk4ß.

We have cloned a novel human mixed-lineage kinase gene, MLK4. Two alternatively spliced forms, MLK4α (580 aa) and MLK4ß (1036 aa), have been identified and mapped to chromosomal band 1q42. MLK4 shows high amino acid homology to the kinase catalytic domain of MLK3 (72%), MLK1 (71%) and MLK2 (69%). Strong expression of MLK4 was detected in the human pancreas and kidneys. pCMV-MLK4ß c-myc-tagged protein (human) was expressed in the cytoplasm and nucleus of transiently transfected COS-1 cells, while pCMV-MLK4α c-myc-tagged protein (human) was expressed in cytoplasm only. Both MLK4 isoforms reduced the colony formation ability of MCF7 cells by 85%-95% and almost totally suppressed cell proliferation in the CyQUANT cell proliferation assay. Human pCMV-MLK4ß transgenic mice expressed the MLK4ß in all tissues examined but no phenotypic abnormalities were observed. Thus, in this work, we present the cloning and sequencing of MLK4α and MLK4ß for the first time; the data obtained suggest that MLK4 may function as a MAP kinase.

Intersectin 1 forms complexes with SGIP1 and Reps1 in clathrin-coated pits.

Intersectin 1 (ITSN1) is an evolutionarily conserved adaptor protein involved in clathrin-mediated endocytosis, cellular signaling and cytoskeleton rearrangement. ITSN1 gene is located on human chromosome 21 in Down syndrome critical region. Several studies confirmed role of ITSN1 in Down syndrome phenotype. Here we report the identification of novel interconnections in the interaction network of this endocytic adaptor. We show that the membrane-deforming protein SGIP1 (Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1) and the signaling adaptor Reps1 (RalBP associated Eps15-homology domain protein) interact with ITSN1 in vivo. Both interactions are mediated by the SH3 domains of ITSN1 and proline-rich motifs of protein partners. Moreover complexes comprising SGIP1, Reps1 and ITSN1 have been identified. We also identified new interactions between SGIP1, Reps1 and the BAR (Bin/amphiphysin/Rvs) domain-containing protein amphiphysin 1. Immunofluorescent data have demonstrated colocalization of ITSN1 with the newly identified protein partners in clathrin-coated pits. These findings expand the role of ITSN1 as a scaffolding molecule bringing together components of endocytic complexes.

Microexon-based regulation of ITSN1 and Src SH3 domains specificity relies on introduction of charged amino acids into the interaction interface.

SH3 domains function as protein-protein interaction modules in assembly of signalling and endocytic protein complexes. Here we report investigations of the mechanism of regulation of the binding properties of the SH3 domains of intersectin (ITSN1) and Src kinase by alternative splicing. Comparative sequence analysis of ITSN1 and Src genes revealed the conservation of alternatively spliced microexons affecting the structure of the SH3 domains in vertebrates. We show that neuron-specific ITSN1 transcripts containing the microexon 20 that encodes five amino acid residues within the SH3A domain are expressed in zebrafish from the earliest stages of the development of the nervous system. Models of alternative isoforms of the ITSN1 SH3A domain revealed that the insertion encoded by the microexon is located at the beginning of the n-Src loop of this domain causing a shift of negatively charged amino acids towards the interaction interface. Mutational analysis confirmed the importance of translocation of these negatively charged amino acids for interaction with dynamin 1. We also identified a residue within the microexon-encoded insert in the SH3 domain of brain-specific variant of Src that abolishes interaction of the domain with dynamin 1. Thus microexons provide a mechanism for the control of tissue-specific interactions of ITSN1 and Src with their partners.

Structural diversity and differential expression of novel human intersectin 1 isoforms.

Intersectin 1 (ITSN1) is an evolutionarily conserved adaptor protein that functions in clathrin-mediated endocytosis, cell signalling and cytoskeleton rearrangements. The ITSN1 gene encodes two main isoforms: a short form (ITSN1-s), which is ubiquitously expressed and consists of two Eps15 homology (EH) domains and five Src homology 3 (SH3) domains, and a long form (ITSN1-l), which is predominantly expressed in the brain and contains three additional domains, a Dbl homology (DH) domain, a Pleckstrin homology (PH) domain and a C2 domain. Using computational analysis of the EST database and 3' RACE we determined the length of the 3' untranslated region of ITSN1-l and demonstrated that the polyadenylation site is located 11,559 nt downstream of the stop codon of the ITSN1-l mRNA. Recently, additional splicing events affecting ITSN1 transcripts were reported, but full-length transcriptional isoforms with different combinations of alternatively spliced exons remained unknown. Here we report the identification of fifteen novel transcriptional isoforms of the human ITSN1 gene with full-length coding sequences that are the result of different combinations of the alternatively spliced exons 5, 6/6', 20, 23, 25, 26, 26a and 35. The isoforms identified differ in domain organization and expression level in different tissues and more likely contribute to the modulation of many complex protein interactions in which ITSN1 participates.