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Human Autoimmune Sera as Molecular Probes for the Identification of an Autoantigen Kinase Signaling Pathway

Human Autoimmune Sera as Molecular Probes for the Identification of an Autoantigen Kinase... 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. apoptosis autoantibodies autoimmunity kinase RNA splicing Footnotes ↵ * Abbreviations used in this paper: CDK, cyclin-dependent kinase; DNA-PK, DNA-dependent protein kinase; MCTD, mixed connective tissue disease; NP40, nonidet P40; RNP, ribonucleoprotein; snRNP, small nuclear RNP; SLE, systemic lupus erythematosus; Sm, Smith complex; SR splicing factor, serine/arginine splicing factor; SRPK, SR protein kinase. Submitted: 7 July 2002 Accepted: 23 September 2002 Revision received 9 September 2002 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Experimental Medicine Rockefeller University Press

Human Autoimmune Sera as Molecular Probes for the Identification of an Autoantigen Kinase Signaling Pathway

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Publisher
Rockefeller University Press
Copyright
© 2002 Rockefeller University Press
ISSN
0022-1007
eISSN
1540-9538
DOI
10.1084/jem.20021167
Publisher site
See Article on Publisher Site

Abstract

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. apoptosis autoantibodies autoimmunity kinase RNA splicing Footnotes ↵ * Abbreviations used in this paper: CDK, cyclin-dependent kinase; DNA-PK, DNA-dependent protein kinase; MCTD, mixed connective tissue disease; NP40, nonidet P40; RNP, ribonucleoprotein; snRNP, small nuclear RNP; SLE, systemic lupus erythematosus; Sm, Smith complex; SR splicing factor, serine/arginine splicing factor; SRPK, SR protein kinase. Submitted: 7 July 2002 Accepted: 23 September 2002 Revision received 9 September 2002

Journal

The Journal of Experimental Medicine – Rockefeller University Press

Published: Nov 4, 2002

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