NEDD9
Neural precursor cell expressed developmentally down-regulated protein 9 is a protein that in humans is encoded by the NEDD9 gene. NEDD-9 is also known as enhancer of filamentation 1, CRK-associated substrate-related protein, and Cas scaffolding protein family member 2. An important paralog of this gene is BCAR1.
Discovery
In 1992, Kumar, et al., first described a sequence tag corresponding to the NEDD9 3′ untranslated region based on the cloning of a group of genes predominantly expressed in the brain of embryonic, but not adult mice, a group of genes designated neural precursor cell expressed, developmentally down-regulated. In 1996, two groups independently described the complete sequence of the NEDD9 gene, and provided initial functional analysis of NEDD9 protein. Law et al. overexpressed a human cDNA library in S. cerevisiae, and screened for genes that simultaneously affected cell cycle and cell polarity controls, inducing a filamentous yeast budding phenotype, and thus identified the HEF1 protein. This study identified HEF1/NEDD9 as an interactive partner for focal adhesion kinase, connecting it to integrin signaling. Separately, Minegishi et al. cloned the gene encoding a protein hyperphosphorylated following ligation of β1-integrins in T cells and hypothesized to play a role in the process of T cell costimulation, designating this gene Cas-L.Gene
The genomic coordinates of the NEDD9 gene are 6:11,183,530-11,382,580 in the GRCh37 assembly, or 6:11,183,298-11,382,348 in the GRCh38 assembly. The gene is on the minus strand. The cytogenetic location is 6p25-p24, based on the nomenclature developed by the Human Genome Organization gene nomenclature committee. NEDD9 is the HGNC approved symbol. Official IDs are 7733, 4739, and ENSG00000111859. CAS-L, CASL, HEF1, dJ49G10.2, dJ761I2.1, CAS2, CASS2 are alias symbols. The NEDD9 gene is conserved in Rhesus monkeys, dogs, cows, mice, rats, chickens, zebrafish, and frogs. In vertebrates, it is a member of a 4-gene family, with the other paralogous genes known as BCAR1, EFS, and CASS4The NEDD9 promoter has 2 transcriptional start sites. The transcript variants NM_006403.3 and NM_001142393.1 encode proteins that have distinct N-termini. In mouse, the two alternative first exons are MKYK and MWAR. Their function is not known. NM_001142393 initiates translation at an upstream location compared to NM_006403.3, but both transcripts have 7 exons. Shorter transcripts with missing exons or an alternative 3' terminal exon have been detected in various studies; however, their role in the cell is unclear.
The 5' region of the NEDD9 promoter is regulated by all-trans retinoic acid, and contains a retinoic acid response element that is specifically bound by a retinoid X receptor /retinoic acid receptor heterodimer. NEDD9 is also induced by the environmental pollutant dioxin, based on regulation through the aryl hydrocarbon receptor. One study has found NEDD9 repressed by estrogen, based on binding of the SAFB1 co-repressor. NEDD9 is induced by Wnt signaling in colon cancer, based on binding to T-cell factor factors in the promoter region. NEDD9 is induced by hypoxia and loss of VHL, based on binding of hypoxia-induced factor transcription factors to the NEDD9 promoter. Prostaglandin E2 induces NEDD9 transcription. The Fox transcription factor Forkhead box C1 and PAX5 transcription factor have been reported to induce NEDD9 transcription. TGF-beta induces NEDD9 transcription. Based on inspection of sequence, the NEDD9 promoter also has potential binding sites for a number of additional transcription factors, including STAT5A and NF-kappa B.
In the 3'UTR of NEDD9 is a match to positions 2-8 of mature miR-145. NEDD9-binding regions in the miR-145 locus would allow the direct binding of the NEDD9 3'UTR to the genomic region of miR-145, and some studies suggests this miR regulates NEDD9 in glioblastoma prostate cancer, and renal cell carcinoma cells. A non-coding RNA, named B2, extending from 10 kb upstream of NEDD9 exon 1 to exon 4 has been described, but the functional role for this ncRNA is not yet clear. NEDD9 is highly expressed in the embryonal brain, and in numerous tissues in the embryo and adult organism. Elevated expression is associated with cancer, as discussed below.
Protein family
NEDD9 is a member of the CAS protein family, which has 4 members in vertebrates. The other paralogous genes are known BCAR1, EFS, and CASS4. There is no detectable NEDD9-related gene in bacteria, yeast, or C. elegans. A single family member exists in D. Melanogaster, termed DCas.Structure
In humans, NEDD9 is 834 amino acids long. NEDD9 is a noncatalytic scaffolding protein that contains docking sites for proteins involved in multiple signal transduction pathways, regulating magnitude and duration of cell signaling cascades The overall structure of NEDD9 is represented graphically in Figure 1.These domains include:
; SH3 domain: This highly conserved N-terminal domain mediates NEDD9 binding to the polyproline motifs of a number of important interacting proteins, with some well-studied partners being FAK and the related PYK2/RAFTK kinase, C3G, PTP-PEST, PTP1B and CIZ.
; Substrate domain : This unstructured region contains multiple YxxP motifs, which are phosphorylated by src family kinases to create binding sites for proteins with SH2 domains, such as Crk. Phosphorylation of these motifs can be activated by mechanical forces such as cytoskeletal stretch. Other phosphorylation events in this region are imposed by the kinase Aurora-A, which phosphorylates residue S296, for processes related to cell cycle control.
; Serine rich region: The SR region likely folds into a 4-helix bundle, based on substantial predicted homology to BCAR1, for which the structure has been solved.
; Focal adhesion targeting domain: The FAT-like C-terminal domain is highly conserved in focal adhesion proteins, and sufficient for localizing focal adhesion kinase to focal adhesions. It forms a four-helix bundle structure and implicated in interaction with NSP proteins, and other proteins such as the Id family of helix-loop-helix proteins.
In terms of post-translational modifications, NEDD9 is subject to significant phosphorylation based on growth conditions. In most actively growing adherent cells, NEDD9 migrates as a doublet of 115 and 105 kDa. Serine/threonine hyper-phosphorylated p115 NEDD9 is more common in G2/M phase cells, suggesting these modifications are associated with increased localization to centrosome and mitotic spindle. One study indicated the conversion of p115 into p105 is activated by cell detachment through cytoskeletal regulation of phosphatase PP2A, although other work has found conflicting results.
Synthesis and degradation
NEDD9 is present throughout cell cycle, but most abundant in G2/M phase cells. NEDD9 is subject to both caspase cleavage and proteasomal degradation. In conditions of cell detachment, and particularly in early stages of anoikis or apoptosis, NEDD9 is rapidly cleaved by caspases 3 and/or 7 at a DLVD site , and at a DDYD site to form N-terminal 55 KDa and C-terminal 28 KDa fragments forms. This cleavage is prevented by focal adhesion formation, which suggests NEDD9 as a sensor of altered adhesion states. Overexpression of p28 in cells causes cellular rounding and detachment, and induces apoptosis, probably because of a dominant-negative effect on survival-promoting signaling complexes at focal adhesions. Together this data suggests that production of different NEDD9 posttranslational modifications is regulated by cell de/attachment, which, in turn, allows regulation of NEDD9 turnover and participation in distinct cellular processes.P115 is the primary target for proteasomal degradation of NEDD9. Proteasomal degradation of NEDD9 is triggered by a number of stimuli, including induction of TGF-beta signaling. An effector of the TGFbeta receptor, Smad3, may interact directly with APC subunit APC10 and thus recruit the APC complex. CDH1 subunit of the APC complex recognizes NEDD9 and regulates ubiquitination and subsequent degradation of NEDD9. NEDD9 is also degraded by the proteasome at the end of mitosis, following completion of activities with Aurora-A that support mitotic progression.
Tissue distribution and intracellular localization
In interphase cells, the majority of NEDD9 localizes to focal adhesions. However, some of the protein is also cytoplasmic, and small pools localize to the centrosome and the basal body of cilia. At mitotic entry NEDD9 moves along mitotic spindle, eventually localizing at midbody at cytokinesis.Function
NEDD9 is an intermediate in a number of important signaling pathways relevant to the cellular processes of proliferation, survival, migration, and others.Integrin, FAK/RAFTK, and SRC kinases
Integrin signaling, which control cell movement, spreading and adhesion to extracellular matrix, and survival, is the best established signaling pathway for NEDD9. Integrins are transmembrane proteins that nucleate focal adhesions, structures that provide bi-directional signaling between ECM and actin cytoskeleton. NEDD9 stabilizes formation and regulates turnover of focal adhesions, influencing cell motility and the invasion and metastasis of cancer cells. In response to integrin activation, FAK or the related kinase RAFTK recruits NEDD9 into a focal adhesion site, binds it via the N-terminal SH3 domain and phosphorylates the NEDD9 Src-binding site. This allows SRC or SRC family kinase to bind NEDD9 via its SH2 domain. Phosphorylation of the NEDD9 substrate domain by Src and other kinases results in the creation of binding sites for Crk and other adaptors that associate with SH2 binding motifs. NEDD9 Crk complexes activate Rho and Ras family GTPases via the recruitment of their nucleotide exchange factors, such as DOCK1, DOCK3 DOCK180 and C3G.These GTPases regulate cell motility, proliferation and also contribute to tumor progression and invasion. In many cell types, NEDD9 overexpression increases spreading and crescent morphology. However, in fibroblasts, some work has found that absence of NEDD9 leads to more rapid focal adhesion turnover, which led to increase of migration in NEDD9-/- compared to wild type.
In cancer cells, NEDD9 can drive mesenchymal-type movement by activating RAC1 GTPase and WAVE in complex with its GEF DOCK3, which in turn cause inhibition of GTPase Rho and amoeboid movement. Invasion is accompanied by proteolysis of the ECM through activation of MMP14, MMP2 and MMP9 metalloproteinases.