Embryonal fyn-associated substrate

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). 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 ==

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. 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). • 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. 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. 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. 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, Upon T-cell receptor (TCR) stimulation, EFS dephosphorylation and release of the SRC family kinase FYN and phospholipase C-γ normally lead to self-limitation of the immune response. Consistent with this mechanism, EFS overexpression in T cell-derived cell lines decreased IL-2 concentration in supernatants in response to TCR stimulation, EFS single nucleotide polymorphisms (SNPs) were subsequently linked to Crohn's disease. SNPs linked to EFS are trans-acting, potentially affecting the level of EFS expression but not its coding sequence. Another study suggested that EFS might contribute to acute rheumatic fever susceptibility. In this work, peripheral blood mononuclear cells (PBMCs) from patients with rheumatoid heart disease (RHD) and control subjects that had never experienced acute rheumatoid fever were stimulated with rheumatogenic and non-rheumatogenic group A streptococci (GAS) strains. EFS was one of only four genes with significantly increased expression in both arms of the study: 1) RHD patient versus control PBMCs after stimulation of both groups with rheumatogenic GAS and 2) RHD patient PBMC stimulated with rheumatogenic versus non-rheumatogenic GAS. Another study has implicated EFS in the Chediak-Higashi syndrome (CHS). and is predicted to result in reduction of gene expression. EFS expression was strongly downregulated in hormonal therapy resistant PC346DCC, PC346Flu1 and PC346Flu2 prostate cancer cells compared to therapy responsive PC346C cells. Another study found that decreased EFS mRNA expression levels are observed in higher Gleason score prostate cancer samples. Low EFS expression also correlated with malignant behavior of the PC-3 and LNCaP prostate cancer cells. In another study, methylation of the EFS CpG island was observed in 69% of cases of uveal melanoma (UM) and only UM with EFS methylation gave rise to metastases. The EFS mRNA was also identified as differentially expressed in two of the three groups of glioblastoma multiforme as identified by gene expression profiles (GEPs). EFS was differentially expressed in the GEP1 and GEP3 groups, which were associated with worse prognosis, with more significant cytogenetic abnormalities and genomic instabilities observed in this groups. At the level of the EFS protein, a study of BT474 breast cancer cells found significant increases in expression of EFS and other proteins relevant to SRC kinase signaling, including CDCP1/Trask and Paxillin, in trastuzumab (Herceptin) resistant versus sensitive cells Importantly, EFS knockdown with siRNA restored trastuzumab sensitivity. Finally, in a 2013 study of castration-resistant prostate cancer, EFS was identified as having significantly increased gross phosphorylation levels in samples from androgen-deprived (AD), long-term AD treated, or castration-resistant prostate carcinoma xenografts, versus in androgen deprivation therapy-naıve xenografts == Clinical significance ==

Based on the above discussion, it is possible that therapeutic benefits can be achieved by using EFS expression or phosphorylation as a marker of disease progression and prognosis in some forms of cancer. Further assessment of EFS expression, mutational status, and potential polymorphic variants may be of use in understanding the biology and developing treatment strategies for immune system pathologies such as CHS. There are currently no therapeutic approaches targeting EFS, and given the protein lacks a catalytic domain and extracellular moieties, it may be challenging to generate such agents. ==Notes==