The Molecular Interaction of Fas and FAP-1

Abstract

Fas (APO-1/CD95) and its ligand have been identified as important signal-mediators of apoptosis (1). The structural organization of Fas (APO-1/CD95) indicates that it is a member of the tumor necrosis factor receptor superfamily, which also includes the p75 nerve growth factor receptor (2), the T-cellactivation marker CD27 (3), the Hodgkin-lymphoma-associated antigen CD30 (4), the human B cell antigen CD40 (5), and T cell antigen OX40 (6). Genetic mutations of both Fas and its ligand have been associated with lymphoproliferative and autoimmune disorders in mice (7,8). Furthermore, alterations of Fas expression level have been implicated in the induction of apoptosis in T-cells infected with human immunodeficiency virus (9). Several Fas-interacting signal transducing molecules, have been identified using yeast two-hybrid and biochemical approaches, including Fas-associated phosphatase-1 (FAP-1) 1 (10), FADD/MORT1/CAP-1/CAP-2 (11-13), and RIP (14). All but FAP-1 associate with the functional cell death domain of Fas and overexpression of FADD/MORT1, or RIP induces apoptosis in cells transfected with these proteins. In contrast, FAP-1 is the only protein that associates with a negative regulatory domain (C-terminal 15 amino acids) (15) of Fas and that inhibits Fas-induced apoptosis.FAP-1 (PTPN13) has several alternatively-spliced forms that are identical to PTP-BAS/hPTP1E/PTPL1 (16 -18), and contains a membrane-binding region similar to those found in the cytoskeleton-associated proteins, ezrin (19), radixin (20), moesin (21), neurofibromatosis type II gene product (NFII) (22), and protein 4.1 (23), as well as in the PTPases PTPH1 (24), PTP-MEG (25), and PTPD1 (26). FAP-1 intriguingly contains six PDZ (GLGF/DHR) domains that are thought to mediate intra-and inter-molecular interactions among protein. The third PDZ repeat of FAP-1 was first identified as a domain showing the specific interaction with the C terminus of Fas receptor (10). In the present study, we first demonstrated that the C-terminal three amino acids (SLV) of human Fas were necessary and sufficient for its interaction with the third PDZ domain of FAP-1. More important, we were able to induce Fas-mediated apoptosis in a colon cancer cell line by the direct cytoplasmic microinjection of this tripeptide (Ac-SLV). MATERIALS AND METHODSConstructions of Libraries and Screenings-To create numerous mutations in a restricted DNA sequence, PCR mutagenesis with degenerate oligonucleotides was employed according to a protocol described elsewhere (27). Based on the homology between human and rat, two palindromic sequences were designed for construction of a semi-random library. The two primers used were 5Ј-CGGAATTCNNNNNNNNNAA-CAGCNNNNNNNNNAATGAANNNCAAAGTCTGNNNTGAGGATC-CTCA-3Ј and 5Ј-CGGAATTCGACTCAGAANNNNNNAACTTCAGA-NNNNNNATCNNNNNNNNNGTCTGAGGATCCTCA-3Ј. Briefly, the two primers (200 pmol each), purified by high pressure liquid chromatography, were annealed at 70°C for 5 min and cooled at 23°C for 60 min. A Klenow fragment (5 units) was used for filling in with a ...

Differential involvement of the CD95 (Fas/APO-1) receptor/ligand system on apoptosis induced by the wild-type p53 gene transfer in human cancer cells

“…Recent studies have identi®ed several molecules related to CD95 signal transduction. Yanagisawa et al (1997) reported that Fas-associated phosphatase-1 (FAP-1) bound to the negative regulatory domain of CD95, leading to the inhibition of CD95-induced apoptosis in DLD-1 cells. This ®nding suggests that the very ®rst step of CD95 signaling is blocked by FAP-1 in DLD-1 cells.…”

Expression of genes that regulate Fas signalling and Fas-mediated apoptosis in colon carcinoma cells

“…The expression of other proteins that interfere with Fas signalling include FAP-1, which is an inhibitory Fas-binding protein that interacts with the Cterminal 15 amino acids of the regulatory domain of the Fas receptor (Sato et al, 1995). FAP-1 has been identified in cultured colon carcinoma cells (Yanagisawa et al, 1997), and its expression was reported to be highest in cell lines and tissues that are relatively resistant to Fas-mediated cytotoxicity (Sato et al, 1995). However, others have shown that cell lines extremely sensitive to Fas-induced apoptosis (HUT78, SKW6.4) expressed high levels of FAP-1, whereas a Fas-resistant line was completely negative for FAP-1 mRNA (Boe R ; Peter et al, 1996).…”

“…reduced expression of the Fas antigen; Moller et al, 1994), or elevated expression of genes that promote cellular survival (Bcl-2; Bedi et al, 1995) or inhibit essential signalling pathways (e.g. FAP-1; Yanagisawa et al, 1997). We have therefore defined the cellular sensitivity to Fas-mediated apoptosis in 10 human colon carcinoma cell lines and have determined the expression of genes known to regulate the function of the Fas signalling pathway in the induction of Fas-dependent apoptosis.…”

“…Cells must also express sufficient levels of all components of the DISC to induce apoptosis. Inhibitory factors that may be expressed include FAP-1 (a Fas-associated phosphatase), which associates with the negative regulatory domain of the receptor to inhibit Fas signalling (Sato et al, 1995), and has been found to be expressed in colon carcinoma cell lines (Yanagisawa et al, 1997). In addition, high-level Bcl-2 expression has also prevented the function of the Fas pathway, and in this regard, decreased anti-Fas sensitivity has correlated with increased expression of Bcl-2 in colorectal carcinomas (Meterissian and Kontogiannea, 1996), and colorectal carcinoma cell lines (Houghton et al, 1997a,b).…”

Live and let die: regulatory mechanisms in Fas-mediated apoptosis

“…FAP-1 is also known as protein-tyrosine phosphatase, nonreceptor-type, 13 (PTNP13) and was found to associate with the carboxy terminal 15 amino acids of human Fas receptor [150]. In vitro inhibition of the interaction between FAP-1 and Fas using synthetic peptides demonstrated that the amino acid motif SLV, found at the carboxy terminus of the Fas receptor, was both sufficient and necessary for binding to FAP-1 [151]. Mouse Fas receptor does not contain this C-terminal motif and does not interact with either mouse FAP-1 (PTP-BL) or human FAP-1 when overexpressed in cells [152].…”