Signal recognition particle and apoptosis
Apoptosis is thought to play an important role in the pathogenesis of several autoimmune diseases. Lupus autoantigens such as Ro, La and the U1-70 kD protein relocalize to cell surface apoptotic blebs following UV-irradiation (Casciola-Rosen et al., 1994), and immunization of mice with either syngeneic apoptotic cells (Mevorach et al., 1998) or apoptotic human T cells (Gensler et al., 1998) leads to the production of specific autoantibody subsets. Posttranslational modifications of proteins during apoptosis have been proposed to be critical to the development of autoantibodies (reviewed in Utz and Anderson, 1998). At least 17 autoantigens are cleaved by caspases (a family of cysteine proteases that cleave target molecules following aspartic acid residues during apoptosis). These autoantigens include the 70 kD component of the U1-small nuclear ribonuclear protein complex (U1-70 kD) (Casciola-Rosen et al., 1994), poly A ribose polymerase (PARP) (Lazebnik et al., 1994), DNA-dependent protein kinase (DNA-PK) (Casciola-Rosen et al., 1995), hnRNP C1 and C2 (Waterhouse et al., 1996), lamins A, B, and C (Lazebnik et al., 1995), the nuclear mitotic apparatus protein (NuMA) (Casiano et al., 1996; Weaver et al., 1996), topoisomerases 1 and 2 (Casiano et al., 1996), the nucleolar protein UBF/NOR-90 (Casiano et al., 1996), and alpha fodrin (Haneji et al., 1997; Marin et al., 1995; reviewed in Utz and Anderson, 1998). Taken together, these observations suggest that apoptotic cells may be uniquely suited to present modified self proteins in such a way that tolerance to these molecules is bypassed.
We have used sera derived from patients with various autoimmune diseases as molecular probes to identify novel apoptosis-related proteins and signal transduction pathways. Using this approach we have discovered a kinase pathway involved in the phosphorylation of an important family of RNA splicing factors during apoptosis (Utz et al., 1997; Utz et al., 1998a). While screening a panel of human autoantisera for their ability to precipitate new phosphoproteins from apoptotic Jurkat cell lysates, we serendipitously identified one serum sample that precipitated a 72 kD phosphoprotein from extracts prepared from untreated Jurkat cells that was no longer observed when extracts were prepared from apoptotic Jurkat cells (Utz et al., 1998b). This serum (serum JB) was derived from a patient with dermatomyositis, and had previously been shown to uniquely immunoprecipitate the signal recognition particle (SRP) (Reeves et al., 1986). Using human and rabbit polyclonal sera raised against individual components of SRP, we have demonstrated that this phosphoprotein is the 72 kD component of SRP (SRP 72). SRP 72, which was not previously known to be a phosphoprotein, is proteolyzed during apoptosis induced my multiple different apoptotic stimuli, but not following a proliferative stimulus such as CD3 cross-linking. SRP 72 is cleaved in its carboxyl terminus, probably following the aspartic acid residue at the sequence 614SELD/A618. This cleavage is inhibited by overexpression of the bcl-2 protein or pretreatment of cells with peptide inhibitors of caspases (e.g., fmk-YVAD and fmk-DEVD, data not shown). SRP 72 is phosphorylated exclusively on serine residues and is cleaved in apoptotic cells derived from all multicellular organisms tested, suggesting that cleavage and phosphorylation of SRP 72 may have functional importance. These preliminary results represent the first description of posttranslational modifications of the SRP autoantigen complex.
SRP is a rare autoantigen target in patients with SLE and dermatomyositis (Okada et al., 1987; Reeves et al., 1986). SRP is a highly conserved cytoplasmic complex composed of a 7S structural RNA molecule and 6 polypeptides, and it mediates the targeting of secretory and integral membrane proteins to the endoplasmic reticulum (ER) (Walter and Blobel, 1980; Walter and Blobel, 1982). The intact particle has at least three separable activities: i.) binding to newly synthesized proteins bearing signal sequence as they emerge from the ribosome; ii.) elongation arrest during translation; and iii.) binding to the SRP receptor, leading to release of elongation arrest and translocation of the targeted protein into the ER lumen. Biochemical mutagenesis experiments of the canine SRP complex have implicated individual domains of SRP in each of these three functions (Siegel and Walter, 1988; Walter and Blobel, 1983). Thus, the 54 kD polypeptide is required for binding to the signal sequence, the 14 kD and 9 kD polypeptides are involved in elongation arrest, and the 68 kD and 72 kD proteins have been implicated in binding to the SRP receptor and promoting the directional translocation of newly translated proteins bearing a signal sequence into the ER lumen. The role played by the 7S RNA molecule is currently unknown. To date, all 6 canine proteins and their yeast homologues (Brown et al., 1994) have been cloned, and the genes encoding human SRP 72 (Utz et al., 1998c) and SRP 14 have also been reported. All 6 yeast SRP genes have been deleted in individual yeast strains (Brown et al., 1994); deletion of each gene prevents the localization of secretory proteins to the ER.
Mutational analysis of canine SRP 68 and SRP 72 has demonstrated that these proteins associate with each other through their carboxyl termini, forming a stable complex with the 7S RNA when subjected to sucrose gradient centrifugation (Lütcke et al., 1993). A 57 kD amino terminal fragment of canine SRP 72 (i.e., a smaller fragment than that generated by caspase cleavage of SRP 72) is still capable of interacting in vitro with SRP 68, while a 42 kD fragment is not (Lütcke et al., 1993). Interestingly, an elastase-generated carboxyl fragment of ~4 kD was observed in this analysis, suggesting that a portion of the carboxyl terminus of SRP 72 is exposed (and by inference, is available for interaction with kinases or regulatory proteins) when associated with other components of SRP (Lütcke et al., 1993).
Targeting of secretory proteins to the ER occurs by a strongly conserved, and presumably highly regulated, mechanism. However, little is known about how this process is governed in eukaryotic cells. In response to exogenous stress such as heat shock, protein translation is regulated by several kinases at an early step (initiation). Examples of such kinases include: mitogen activated protein kinases (MAP kinases), which repress translation by phosphorylating the mRNA cap-binding protein eIF4E (Sonenberg and Gingras, 1998); the interferon-inducible, dsRNA-regulated protein kinase (PKR), which inhibits translation by phosphorylating the eIF2 initiation factor (Clemens, 1997; de Haro et al., 1996); and the rapamycin-sensitive p70 S6 kinase, which modulates translation through phosphorylation of the S6 component of ribosomes (Proud, 1996). The observation that SRP 72 is phosphorylated on serine residue(s) in all cell types tested suggests that a serine kinase and/or phosphatase may regulate gene expression at a later step than the translation initiation checkpoint that is regulated by the above kinases. Moreover, since the carboxyl terminus of SRP 72 (which is likely to harbor the serine phosphorylation site) is removed from SRP 72, We hypothesize that SRP containing truncated SRP 72 may be dysregulated during apoptosis. Characterization of the phosphorylation site(s) in the carboxyl terminus of SRP 72, and ultimately the relevant kinase, may elucidate the role this posttranslational modification plays in endoplasmic reticulum trafficking.
Caspase-mediated cleavage of certain proteins during apoptosis has been shown to contribute directly to the apoptotic phenotype. For example, cleavage of p21-activated kinase (PAK2) is required for the formation of apoptotic bodies (Rudel and Bokoch, 1997), and cleavage of gelsolin is responsible in part for cell rounding and nuclear fragmentation, both of which are characteristic features of cell death (Kothakota et al., 1997). Modifications of SRP components during apoptosis may similarly have at least 3 important physiologic consequences. First, SRP 72 cleavage may play a critical role in the execution phase of cell death when apoptosis is initiated by enveloped viruses, since many viral proteins pass through the ER. This would be thwarted if SRP was no longer capable of targeting nascent viral proteins to the SRP receptor on the outer lumen of the ER, and would suggest that SRP 72 cleavage may protect neighboring cells by preventing viral replication and shedding. Second, SRP 72 cleavage may contribute to apoptosis induced by growth factor deprivation. Most growth factors require ER processing prior to secretion, and destruction of this pathway could lead to the inability of dying cells to secrete required growth factors, thus inducing apoptosis of neighboring cells via autocrine or paracrine mechanisms. Finally, inhibition of ER transit during apoptosis may prevent release of inflammatory cytokines such as interleukins or tumor necrosis factor, thus preventing an unwanted and unchecked inflammatory response to cellular debris produced by dying cells.
In this project we are testing the hypothesis that phosphorylation and cleavage of SRP 72 alter the ability of the intact SRP autoantigen complex to target secretory proteins to the ER.
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These studies were funded by an Arthritis Investigator Award (PJU), CCIS
(FC), and a CONACYT Fellowship from the Mexican government (MV).
