Apoptosis Signaling and Autoimmunity
Apoptosis is a morphologically and biochemically distinct form of cell death that occurs in response to a diverse range of stimuli, including irradiation and activation of death receptors such as Fas and the tumor necrosis factor receptor (TNFR) (1). Defects in apoptotic cell death have been linked to autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and scleroderma (reviewed in 2, 3). Since the initial demonstration by Casciola-Rosen and colleagues that SLE autoantigens are clustered in cell surface apoptotic blebs following UV irradiation (4), there has been great interest in elucidating the role played by apoptosis and other forms of cell death in the pathogenesis of systemic autoimmune diseases (5, 6). In addition to relocalization of autoantigens to apoptotic blebs, there is now ample evidence that modifications of autoantigens during apoptosis may also be involved in the genesis of autoantibodies in several diseases, particularly SLE (2, 7). For example, many autoantigens are cleaved in vivo and in vitro by a family of proteases (caspases) at sites containing an aspartic acid residue at the P1 position (8, 9). Another protease, the cytotoxic granule protein granzyme B, has recently been shown by Casciola-Rosen and colleagues to uniquely cleave most autoantigens at sites that are not recognized by caspases (10, 11). Other modifications of autoantigens such as DNA cleavage (12), DNA methylation (13), RNA cleavage (14-17), protein transglutamination (18), citrullination (19), and ubiquitination (20) have also been associated with cell death (reviewed in 2, 3). This largely observational body of evidence is now supported by in vivo immunization studies in which mice immunized with syngeneic (21) or human (22) apoptotic cells develop antibodies recognizing autoantigens such as Ku, U-snRNPs, and ribosomes (23-26).
SR proteins comprise a family of pre-mRNA splicing factors that regulate constitutive (i.e., splicing that occurs at the same site in a given RNA molecule in every cell) and alternative mRNA splicing (i.e., splicing that occurs at different sites in the RNA in some or all cells) (27-28). The splicing activity of SR proteins is modulated by addition of phosphate groups to their SR domains (28). Interestingly, autoantibodies directed against SR proteins have been identified in the serum of SLE patients, and the recognition by antibodies occurs in a phosphorylation-dependent manner (29). To date, 6 kinases have been described that possess SR protein kinase activity. One of these proteins is the scleroderma autoantigen Scl-70, which surprisingly possesses a protein kinase activity (27). Purified topoisomerase I phosphorylates several serine arginine (SR) splicing factors including ASF/SF2 and SC35, and the kinase activity is inhibited by addition of the topoisomerase inhibitor camptothecin in the presence of DNA (27). In addition to Scl-70, at least 5 other serine arginine protein kinases (SRPKs) have been described. These include SRPK1, SRPK2, and Clk/Sty 1, 2 and 3 (30-33).
The observation that many autoantigens are cleaved by caspases led our lab to use human sera derived from patients with autoimmune diseases to identify proteins that are phosphorylated by kinases during apoptosis (34-36). These experiments led to the discovery of a novel kinase pathway involved in the phosphorylation of SR proteins during apoptosis (37, 38). We have demonstrated that several different SR proteins associate with both the U1-snRNP (a lupus and MCTD autoantigen) and the U3-snoRNP (a scleroderma autoantigen) during apoptosis, implicating this signaling cascade in the subsequent development of disease-specific autoantibodies (37-39). A main goal of this project is to identify the kinase pathways responsible for generating phosphorylated SR splicing factors.
The apoptosis threshold that must be overcome in a given cell is controlled by multiple mechanisms. First, relative levels of death-inducing molecules (e.g., Ich-1L and bcl-xS) and protective molecules (e.g., bcl-2 and bcl-x L) set a threshold that must be overcome for a given stimulus to irreversibly activate the cell death pathway (44-46). Second, stressed cells can respond to stimuli by activating kinase pathways, ultimately converging in the nucleus where activation of protective genes such as IEX-1 occurs (47-49). Third, it is now widely accepted that apoptosis can be regulated at the level of mRNA splicing. The RNA splicing reaction can generate several different forms of a given molecule with opposite functions by a process termed "alternative RNA splicing." For example, it has been shown that cells expressing the larger splice variant of the bcl-x gene (bcl-x L) are protected against cell death, while cells expressing the short form (bcl-xS) have an increased susceptibility to cell death (44-46, 50). Similar regulation has been described for the C. elegans ced-4 gene product (51), for the death domain-containing receptor LARD (52), and for the death protease caspase 2 (Nedd2/Ich1) (53). Overall, more than 30 apoptosis effector molecules that are regulated at the level of alternative mRNA splicing have been described (54). However, it is unknown how levels of the opposing factors are determined, nor is it known whether relative levels of antagonistic factors can fluctuate in response to external stimuli such as irradiation or immunosuppressive agents. We are testing the hypothesis that activation of an SR protein kinase pathway during cell death might play an important role in determining cell death thresholds, alternative splicing of mRNAs, and autoimmunity.
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Funding for this project was provided by an NIH K08 Award (PJU) and a Stanford University Dean's Fellowship (MK).
