Embryonal fyn-associated substrate is a protein that in humans is encoded by the EFS gene. It is also known as CASS3.
History and discovery
EFS (Embryonal Fyn-associated Substrate), also known as SIN (Src INteracting or Signal Integrating protein) was originally identified using cDNA library screening of mouse embryonal libraries for proteins containing SH3-interacting domains, or interacting with the SRC SH3 domain, in two independent studies by Ishino et al. in 1995 and Alexandropoulos et al. in 1996.
In humans, the 561 amino acid EFS protein acts as a scaffolding protein for cell signaling based on interactions with SRC, FAK, and other proteins, and has been linked to roles in the function of the immune system, and the development of cancer.
Gene
The chromosomal location of the EFS gene is 14q11.2 and its genomic coordinates are 14:23356400-23365633 on the reverse strand in GRChB38p2 (Genome Reference Consortium Human Build 38 patch release 2). According to the Human Genome Organization (HUGO) Gene Nomenclature Committee (HGNC) its approved symbol is EFS and its synonyms are "Cas scaffolding protein family member 3", CASS3, EFS1, EFS2, HEFS and SIN. The official Gene IDs assigned to EFS are 16898 (HGNC), 10278 (Entrez Gene) and ENSG00000100842 (Ensembl).
In humans, at least three transcript variants are known for EFS: isoform 1, containing 6 exons end encoding the full-length protein with 561 amino acids; isoform 2, containing 5 exons and encoding a shorter protein (468 amino acids in length); and isoform 3, containing 6 exons and encoding the shortest protein (392 amino acids).
Little is known about the transcriptional regulation of EFS, but several transcriptional regulators for EFS have been proposed based on consensus binding sites in its promoter region for ATF (Activating transcription factor), NF-κβ, NF-κβ1, GATA-3, C/EBPα (CCAAT/enhancer-binding protein alpha), glucocorticoid receptors α and β, and p53. Expression of isoforms 1 and 2 has been detected in multiple tissues, with maximal expression in the placenta, and the embryonal central nervous system, heart, testes and lungs. Although its expression has been reported as lower in thymus and lymphocytes, functional studies of EFS to date have best defined it as important for immune system function. One screen for implantation-related genes regulated by progesterone found that EFS was downregulated by 17β-estradiol and progesterone in explants of late proliferative phase endometrium.
Protein family
EFS is a member of the CAS (Crk-Associated Substrate) family of proteins. In humans and mammals, this group consists of four members: p130Cas/BCAR1, NEDD9/HEF1, CASS4 and EFS. There are no paraloguous genes for this family in yeasts and fungi, diploblasts and nematodes such as C. elegans. A single ancestral member is found in Drosophila.
Structure +Table 1. EFS structure.Domain Position Length Function N-terminal 1 - 44 aaThis region has no assigned function SH3-domain 5-6864 aaBinds to proline-rich motif containing proteins, such as FAK, PTK2B, C3G, PTP-PEST, PTP1B, CIZ and FRNK. SH2-binding region 69 - 350282 aaContains YxxP motifs capable of being phosphorylated on tyrosine residues, then binding SH2 domains. Serine rich domain 351 - 488138 aaConserved domain structure encompassing 4 α-helices bundle has a docking function. C-terminal 489 - 56173 aaConserved domain structure encompassing 4 α-helices bundle has a docking function; homo- or heterodimerization; focal adhesion targeting. As the member of CAS protein family, EFS shares common structural characteristics with other members of the family. This includes 4 defined domains (summarized in Table 1): An N-terminal SH3 domain that is highly conserved among the 4 CAS family members, and highly conserved throughout evolution (amino acids 5-68 for human EFS). SH3 domains bind to proline-rich motif containing proteins. The amino acid sequences of the SH3 domains are 70% identical among human EFS, BCAR1, and NEDD9, making this the most highly conserved domain for the whole protein family. Notably, the murine and human EFS SH3 domains are 100% identical, while the rest of the amino acid sequences of mouse and human EFS are only 78% identical. Important binding partners for this region include FAK, PTK2B, C3G, PTP-PEST, PTP1B, CIZ, and FRNK. Central "substrate domain" containing multiple repeats of tyrosine residues embedded within specific conserved sequences (YxxP) (amino acids 69-350 for human EFS). This region contains 9 such binding sites, in contrast to family members BCAR1 and NEDD9 (20 and 18 motifs, respectively) and similar to CASS4 (estimated at 10 such motifs). Once phosphorylated by SRC or other kinases, these tyrosine motifs are bound by the SH2 domains of signaling proteins. Important binding partners for this region include Crk1/2 and Crk-L, a Crk1 paralogue. A serine-rich domain encompassing a 4 α-helix bundle (amino acids 351-488 for human EFS). Although primary amino acid sequence shows considerable divergence versus other CAS family members in this region, structural analysis predicts that this bundle has a highly conserved fold and provides a docking site for family members. A C-terminal domain (489-561 amino acids in human EFS) is highly conserved between family members at both primary amino acid sequence and predicted fold. All CAS proteins except CASS4 contain a YDYVHL motif within this domain, which is an important binding site for the Src SH2 domain. This region is considered to possess a homo- or heterodimerization ability. There are three protein isoforms of human Efs. hEfs1 and hEfs2 were identified by Ishino et al. hEFS1 (561 aa) represents the human counterpart of mouse embryonal Efs (mEfs1) originally identified. hEFS1 and mEfs1 are 80% identical in their amino acid sequences and 100% identical within the SH3 domain. hEFS2 (468 aa) is identical to hEFS1, except for its lack of the SH3 domain. hEFS3 (392 aa) also lacks a functional SH3 domain and has the same C-terminus and short N-terminal amino acid tail as the full-length protein. Although little functional analysis of hEFS2 has been performed, speculatively, given lack of an SH3 domain, abundant hEFS2 may inhibit hEFS1 signaling by titrating partner proteins. As of 2015, there has been no functional analysis of hEFS3.
Function
As a member of the CAS protein family, EFS is a multi-domain docking molecule that lacks any known enzymatic activity, but instead mediates signaling by promoting protein–protein interactions through conserved sequence motifs (Figure 1).
An important role of EFS as a CAS-family member function is transmission of integrin-initiated signals from the extracellular matrix to downstream effectors, leading to reorganization of the actin cytoskeleton and changes in motility and invasion. The SH3 domain is a point of contact with polyproline sequences on focal adhesion kinase (FAK). or the related kinase PTK2B, also known as RAFTK/Pyk2/CAKβ. Typically, phosphorylation of the C-terminal region of CAS proteins by FAK or PTK2B creates a binding site for the SH2 domain of a SRC-family protein, which then hyper-phosphorylates the substrate domain, allowing the CAS protein to function as a scaffold for other proteins including CRK proteins and C3G, a guanine nucleotide exchange factor (GEF) for RAP1. PTP-PEST, a soluble protein tyrosine phosphatase that is ubiquitously expressed in mice both during embryonic development and in adult tissues, opposes FAK and PTK2B activity, as it dephosphorylates PTK2B, FAK and CAS family members, among other proteins. The PTP-PEST proline-rich sequence 332PPKPPR337 has been shown to interact directly with the SH3 domain of members of EFS and another CAS protein, NEDD9.
In normal untransformed cells, EFS acts as a SRC family kinase substrate in neurite outgrowth, a process that is dependent on activity of SRC kinases. Reciprocally, EFS activates SRC signaling through c-CRK and RAP1. Further, SRC directly phosphorylates residues Y576 and Y577 tyrosine sites on the EFS, enhancing targeting FAK, and eventually the solubility and/or stability of the complex. Through SRC, EFS may also negatively regulate expression of E-cadherin at adherens junctions, a function that has been reported for other CAS proteins (NEDD9 and BCAR1); however, this point has not been directly established for EFS.
Disease association
The well-studied CAS proteins BCAR1 and NEDD9 have important roles in cancer and other pathological conditions, which have been addressed in many studies and reviews. EFS has attracted less study. However, the conserved functional properties of EFS relevant to cellular adhesion and migration, and RTK signaling, suggest changes in activity of this protein may also be relevant to cancer and other disease states, influencing prognosis and therapeutic response. The changes in EFS expression and post-translational modification in the context of disease discussed below are summarized in Table 2. Disease Study finding for EFS Crohn's diseaseThe study linked EFS gene to Crohn's disease (p-value 0.039) in humans. Rheumatic fever susceptibility Significantly increased expression after stimulation of peripheral blood mononuclear cells from patients with rheumatoid heart disease. Prostate cancerCpG site hypermethylation of EFS was associated with prediction of biochemical, local, and systemic recurrence of prostate cancer. Decreased EFS expression was shown in advanced prostate cancer compared to normal tissue, which correlated with high metastatic potential.Uveal melanomaHigh frequency of promoter CpG site methylation and association with a higher risk of metastatic progression. HER2+ breast cancerEFS may play a role in trastuzumab resistance mechanism.ProlactinomaEFS may be involved in stem cell regulation, tumor cell invasion, tumor recurrence, and drug resistance. Gestational choriocarcinomaLocated in a frequently amplified chromosomal region along with >100 other genes. Glioblastoma multiformeOne of the genes differentially expressed in two sub-groups of glioblastoma multiforme defined by gene expression profile. Chediak-Higashi syndromeDirect interaction with LYST protein, which is associated with lysosomal trafficking. Human endometrium expression profilingDown regulated by 17β-estradiol and progesterone in explants of late proliferative phase endometrium.
Role in inflammation and T-Cell function
EFS regulates T-cell function and maturation, preventing expansion of autoreactive clones and pathological immune responses. Two studies that have reported that EFS expression in medullar thymus epithelial cells is important for negative selection of T-cells during their development, which implies an important role of EFS in maintaining immune homeostasis and autoimmunity prevention. In these studies, mice with defective EFS progressed normally during embryogenesis but then developed massive inflammatory lesions in multiple tissues that bore a striking histological resemblance to inflammatory bowel diseases such as Crohn's disease. Mechanistically, EFS expressed in medullary thymic epithelial cells (mTECs) is crucial for their functional maturation and growth factor-mediated expansion. mTECs are important for proper T-cell maturation and negative selection of autoreactive clones, required for development of immunological self-tolerance.