Human autoimmune sera as molecular probes for the identification of an autoantigen kinase signaling pathway.
Using human autoimmune sera as molecular probes, we previously described the association of phosphorylated serine/arginine splicing factors (SR splicing factors) with the U1-small nuclear ribonucleoprotein (U1-snRNP) and U3-small nucleolar RNP (snoRNP) in apoptotic cells. SR proteins are highly conserved autoantigens whose activity is tightly regulated by reversible phosphorylation of serine residues by at least eight different SR protein kinase kinases (SRPKs), including SRPK1, SRPK2, and the scleroderma autoantigen topoisomerase I. In this report, we demonstrate that only one of the known SRPKs, SRPK1, is associated with the U1-snRNP autoantigen complex in healthy and apoptotic cells. SRPK1 is activated early during apoptosis, followed by caspase-mediated proteolytic inactivation at later time points. SRPKs are cleaved in vivo after multiple apoptotic stimuli, and cleavage can be inhibited by overexpression of bcl-2 and bcl-x(L), and by exposure to soluble peptide caspase inhibitors. Incubation of recombinant caspases with in vitro-translated SRPKs demonstrates that SRPK1 and SRPK2 are in vitro substrates for caspases-8 and -9, respectively. In contrast, topoisomerase I is cleaved by downstream caspases (-3 and -6). Since each of these SRPKs sits at a distinct checkpoint in the caspase cascade, SRPKs may serve an important role in signaling pathways governing apoptosis, alternative mRNA splicing, SR protein trafficking, RNA stability, and possibly the generation of autoantibodies directed against splicing factors.
Kamachi
Makoto
M
Department of Medicine, Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Le
Truc M
TM
Kim
Susan J
SJ
Geiger
Meghan E
ME
Anderson
Paul
P
Utz
Paul J
PJ
D017209:Apoptosis; D001323:Autoantibodies / Q000097:blood / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology*; D020169:Caspases / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004789:Enzyme Activation; D015870:Gene Expression; D006801:Humans; D019169:Jurkat Cells; D008180:Lupus Erythematosus, Systemic / Q000097:blood / Q000276:immunology*; D008947:Mixed Connective Tissue Disease / Q000097:blood / Q000276:immunology*; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000276:immunology* / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000276:immunology*; D015398:Signal Transduction / Q000276:immunology*; D014407:Tumor Cells, Cultured; D051020:bcl-X Protein; D019014:fas Receptor / Q000378:metabolism
D001323:Autoantibodies; D001324:Autoantigens; C494811:BCL2L1 protein, human; D019253:Proto-Oncogene Proteins c-bcl-2; D017412:Ribonucleoprotein, U1 Small Nuclear; D051020:bcl-X Protein; D019014:fas Receptor; C087728:SRPK1 protein, human; D017346:Protein Serine-Threonine Kinases; C110522:SRPK2 protein, human; D020169:Caspases; D004264:DNA Topoisomerases, Type I