In a yeast two hybrid screen we were able to identify several OSTL-interacting proteins from a Hela-cDNA bank (Figure 6.22). Interestingly, two OSTL-interacting proteins are involved in B-cell survival and B-cell receptor signaling.
HAX-1 (HS1-associated protein X-1) was first described to interact with HS1 in B-cells (Suzuki, 1997). HS1 (hematopoietic lyn substrate 1) is a component of the B-cell receptor (BCR) signaling pathway, its expression restricted to hematopoietic cells and it is rapidly tyrosine phosphorylated by a variety of non-receptor tyrosine kinases after lymphocytes stimulation (Kitamura, 1989; Yamanashi, 1993; Brunati, 1995). The activity of HS1 and possibly HAX1 may be important for survival of lymphocytes (Taniuchi, 1995; Suzuki, 1997). Suzuki and coworkers found that the HAX1 open reading frame encoded a protein consisting of 279 amino acids (Figure 7.2 A) with a calculated molecular mass of 35 kDa. The HAX1 protein is hydrophilic, but contains a presumptive membrane-spanning hydrophobic domain at its carboxy terminus.
HAX1 as a whole has no significant homologies to other proteins, but shows some similarity to Nip3, which has been reported to interact with the adenovirus E1B and Bcl-2 protein (Boyd, 1994). HAX-1 possesses two Bcl-2 homologous domains BH1 and BH2 that are conserved among the Bcl-2 family proteins and are known to be critical for the regulation of apoptosis (Figure 7.2 A) (Yin, 1994; Suzuki, 1997). HAX1 contains a PEST sequence, suggesting that this protein may be degraded rapidly (Rogers, 1986) (Figure 7.2 A). Its mRNA is found in all tissues suggesting an essential function of this gene in intracellular signaling (Suzuki, 1997). The HAX1 protein has been reported to localize to the mitochondria, cytoplasm or plasma membrane (Suzuki, 1997; Gallagher, 2000; Dufva, 2001; Sharp, 2002).
HAX1 interacts with the polycystic kidney disease protein (PKD2) and this interaction probably mediates its association with the actin cytoskeleton (Gallagher, 2000).
Interaction between HAX1 and Epstein-Barr virus nuclear antigen leader protein (EBNA-LP or EBNA5) was demonstrated by Kawaguchi, 2000 and by Dufva, 2001 and again suggesting a role of HAX1 in apoptosis.
Recently a model of complex formation was proposed for the EBNA-LP, HAX1 and Bcl-2 proteins. EBNA-LP interacts with HAX1 via its nuclear localization signal (NLS) and HAX1 associates with Bcl-2 through its BH2 domain, further supporting the hypothesis that HAX1 is a regulator of apoptosis (Matsuda, 2003). Yin and coworkers in 2001 reported interaction between HAX1 and IL-1α and suggested that HAX1 may modulate multiple facets of the N- terminal peptide of interleukin-1α (IL-1 NTP) biochemistry.
Through interaction between HAX1 and Kaposi’s Sarcoma-associated herpesvirus protein (K15), Sharp and coworkers in 2002 were able to show that HAX1 is able to block BAX induced apoptosis (BAX is a Bcl-2 family member with a strong proapoptotic function). HAX1 was found to be upregulated in psoriatic lesions (Mirmohammadsadegh, 2003). Mirmohammadsadegh and coworkers analyzed UBV-induced apoptosis in HaCaT keratinocytes overexpressing HAX1 mRNA or its antisense and found further evidence for the role of HAX1 in antiapoptotic processes.
HAX1-specific antisense led to a marked increase in caspase-3 mediated apoptosis, strongly suggesting an antiapoptotic role for HAX1 (Mirmohammadsadegh, 2003). HAX1 interacts with bile salt export protein (BSEP) and regulates its abundance in the apical membrane of Madin-Darby canine kidney cells (Ortiz, 2004). A mechanism was proposed suggesting HAX1 involvement in clathrin-mediated endocytosis: BSEP and HAX1 are present in clathrin-coated vesicles; expression of dominant negative EPS15, which selectively blocks
clathrin mediated endocytosis (Benmerah, 1999), doubled the apical membrane concentration of BSEP; and expression of dominant negative cortactin also doubled the amount of BSEP in the apical membrane. Cortactin, an actin and HAX1-binding protein, participates in clathrin endocytosis (Lynch, 2003; Cao, 2003).
HAX1 interacts with Gα13, the α-subunit of the heterotrimeric G protein G13, this protein has
been shown to stimulate cell migration in addition to inducing oncogenic transformation (Radhika, 2004).
HAX1 potentiate activation of Rac through Gα13; Rac stimulates the translocation of cortactin
(Patel, 1998; Weed, 1998); cortactin stimulates actin polymerization leading to lamellipodia formation (Weed, 1998; Uruno, 2001; Uruno, 2003); and HAX1 promotes Gα13-mediated cell
motility, suggesting that Gα13 and HAX1 are part of a signaling complex involved in cell
motility (Radhika, 2004).
Finally, HAX1 interacts with Omi/HtrA2, a nuclear-encoded mitochondrial serine protease that has a pro-apoptotic function in mammalian cells (Cilenti, 2004). Cilenti and coworkers could show that through this interaction, Omi cleaves HAX1 and can initiate apoptosis from mitochondria.
In our work we could show interaction between OSTL and HAX1 and this interaction was confirmed by using in vivo and in vitro assays. Interestingly, by using OSTL as bait in a yeast two hybrid assay we were able to identify eight different HAX1 clones (Figure 6.25). We selected three clones (2, 3, and 5) and confirmed the interaction by cotransformation assay in yeast (Figure 6.26). Further assays to confirm the interaction between OSTL and HAX1 were performed using HAX1 clone 5, which contains almost the whole HAX1 cDNA (Figure 6.25). The interaction was mapped by cotransformation between different Ostl mutants with HAX1 clone 5 (Figure 6.29). The OSTL variant RING1 (the N terminal RING finger of OSTL) seems to be involved in the interaction with HAX1 (Figure 6.29).
Surprisingly, Ostl mutants 1 and 3 (lanes 2 and 4 in figure 6.29) did not show any interaction with HAX1 (any growth in the yeast cotransformation), although they have only one amino acid that is different from the Ostl mutant 2 and human OSTL (lanes 3 and 8 in figure 6.29) which are capable of interaction with HAX1 (they showed a very nice growth in the yeast cotransformation assay). In Ostl mutants 2 and 4 a serine (S) at position 2 is substituted by a adenine (A), this substitution might be responsible for the difference observed in the interaction assay (Figure 6.29).
in the cytoplasm of the mouse fibroblast cells. Colocalization was also observed in the mitochondria (data not showed).
The in vitro confirmation of interaction was by coimmunoprecipitation using S35 labeled myc- GAL4DBD-OSTL and HA-GAL4AD-HAX1 proteins. Both proteins could be coprecipitated using an anti myc antibody (Figure 6.35).
CFP-OSTL and YFP-HAX1 overexpressed in 293 cells could be coprecipitated (Figure 6.36). This assay confirmed in vivo the association of the two proteins.
Figure 7.2: A) Schematic representation of HAX1 amino acid (aa) sequence and domains. BCL-2 homology domains 1 and 2 (BH1 and -2), a PEST domain, and a transmembrane domain (TMD) are indicated (Figure adapted from Sharp, 2002). B) Schematic representation of Siva-1 and Siva-2 aa sequences. A death domain homology region (DDHR), and a cysteine-rich domain are represented in Siva-1 diagram. Siva-2 lacks the exon 2, corresponding to the 65 aa of the DDHR (Figure adapted from Py, 2004).
SIVA (from Shiva, the Hindu god of destruction) was first described to bind to the CD27 cytoplasmic tail in a yeast two hybrid assay and function as a proapoptotic protein (Prasad, 1997). The human SIVA protein contains 189 aa (Prasad, 1997), the rat Siva 177 aa. Human and rat SIVA are 92% homologous (Padanilam, 1998). Analysis of the primary amino acid sequence revealed an amino-terminal region that has a 40% homology to the death domains (DDs) of FADD and RIP. The homologies between SIVA and TRADD/Ankyrin DDs is 16% and between SIVA and FADD DDs 14% (Yoon, 1999). SIVA has a carboxy-terminal region
rich in cystein residues that could form a B-box-like ring finger (Yang, 1997) (Figure 7.2 B). The B-box region of SIVA, however, lacks any histidine residues. The amino-terminal RING finger and the carboxy-terminal coiled-coil domain, which are characteristic of other B-box- containing proteins (Yang, 1997), are absent in SIVA. Instead, SIVA has additional cystein residues at the carboxy terminus that can potentially form a zinc finger (residues 164-184 in the human SIVA).
Alternatively, the Cys-rich region of SIVA could represent a novel metal binding motif involved in either protein-protein or protein-DNA interactions. The architecture of SIVA is unlike that of any other protein known to bind to the cytoplasmic tails of TNFR family members. The human SIVA mRNA is expressed mainly in thymus and testis, but also in spleen, prostate, ovary, small intestine, PB and to a lesser amount in colon. The SIVA expression was also observed in some cell lines including HL60, HeLa, Raji, K562, MOLT4, SW480, and G361 (Prasad, 1997). In 1997 Prasad and coworkers could demonstrate the proapoptotic properties of SIVA in various cell lines (293, Jurkat, Raji, a murine pre-B cell (SKW), and Ramos) by transiently expressing SIVA as a GFP fusion protein.
In 1999, Yoon and coworkers showed that mouse SIVA is 80% homologous to human SIVA. The murine Siva mRNA is mainly expressed in testis, muscle, and heart with brain expressing the least. Two forms of mouse Siva cDNA were identified by Yoon and coworkers. The longer form was named Siva-1 and the shorter form Siva-2 (Yoon et al., 1999) (Figure 7.2 B). Part of the amino terminal region and most of the death domain homology region (DDHR) including the conserved 13 amino acids stretch is missing in Siva-2. Although Siva-2 lacks the major portion of the DDHR, it showed association with the cytoplasmic tail of the CD27 receptor, as did Siva-1 (Figure 7.2 B). However, murine and human SIVA-2 are much less proapoptotic than SIVA-1 (Yoon, 1999). SIVA was also reported to be up-regulated in colorectal cancer (Okuno, 2001) and research groups have recently reported the apoptotic activity of SIVA (Lin, 1999; Xiao, 2000; Henke, 2000; Cao, 2001; Qin, 2002; Xue, 2002; Spinicelli; 2002; Daoud, 2003; Chu, 2004; Seseke, 2004; Py, 2004; Fortin, 2004; Novikowa, 2005).
We found interaction between OSTL and the proapoptotic human SIVA-1 in our yeast two hybrid assay (Tables 6.6 and 6.8; Figure 6.25; and attachment 4). We were able to confirm the interaction through in vivo (Figure 6.33, 6.37) and in vitro assays (Figure 6.26, 6.30). The RING finger at the N terminal region of OSTL (variant RING1) and the C terminal region of OSTL seems to be involved in the interaction between OSTL and SIVA (Figure 6.30).
The mapping of OSTL-SIVA interaction showed the same pattern of yeast growth that was observed for the OSTL-HAX1 interaction mapping. The amino acid substitutions (V→Q), is probably responsible for the negative interaction results between Ostl mutants 1 and 3 and SIVA (Figures 6.29 and 6.30).
CFP-OSTL and YFP-SIVA overexpressed in 293 cells could be coprecipitated (Figure 6.37). This assay confirmed in vivo the association of the two proteins.
Summarizing our yeast two hybrid data, we propose a functional pathway for OSTL through its interaction with HAX1 and SIVA (Figure 7.3). SIVA and HAX1 are involved in pro- and antiapoptotic decisions in the cell, respectively.