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Human autoimmune sera as molecular probes for the identification of an autoantigen kinase signaling pathway.

Kamachi M, Le TM, Kim SJ, Geiger ME, Anderson P, Utz PJ - J. Exp. Med. (2002)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, CA 94305, USA.

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.

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Human SRPK1 and SRPK2 are differentially cleaved by recombinant caspases in vitro. SRPK1 and SRPK2 were synthesized by in vitro transcription/translation and incubated with the recombinant caspases indicated by number at the top of each figure. Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and detected by autoradiography. The relative migration of molecular size markers in kilodaltons is indicated on the left side of each panel. Lanes are numbered at the bottom of each panel.
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fig3: Human SRPK1 and SRPK2 are differentially cleaved by recombinant caspases in vitro. SRPK1 and SRPK2 were synthesized by in vitro transcription/translation and incubated with the recombinant caspases indicated by number at the top of each figure. Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and detected by autoradiography. The relative migration of molecular size markers in kilodaltons is indicated on the left side of each panel. Lanes are numbered at the bottom of each panel.

Mentions: Previous studies have demonstrated that an SRPK (topoisomerase I) is cleaved by caspases-3 and -6 at unconventional sites during apoptosis (23). The results shown in Fig. 1 further suggested that another SRPK (SRPK1), and perhaps other SRPKs, may also undergo proteolysis during apoptosis. We tested this hypothesis by using a well-validated in vitro cleavage assay using recombinant caspases. In vitro–transcribed/translated SRPKs were incubated in caspase cleavage buffer in the presence of recombinant caspases or a control bacterial lysate, separated by SDS-PAGE and analyzed by autoradiography. As shown in Fig. 3, A and B , human SRPK1 and SRPK2 are differentially cleaved by recombinant caspase-8 and caspase-9, respectively, yielding signature ∼66-kD and 34-kD fragments (SRPK1) and 80-kD and 50-kD fragments (SRPK2), respectively. None of the other caspases tested had the ability to cleave SRPK1 and SRPK2, although caspase-1 appeared to partially cleave human SRPK1, generating fragments of identical size to those observed with caspase-8 (lane 2). Mouse SRPK1 and SRPK2, which are 92 and 58% conserved between mouse and man, respectively, were also differentially cleaved by caspase-8 and caspase-9, respectively, generating fragments of approximately the same molecular weight (unpublished data). In contrast, none of the Clk/Sty isoforms was cleaved by recombinant caspases (unpublished data). Taken together with the published data on topoisomerase I, these results demonstrate that at least 3 of the 8 known SRPKs are differentially cleaved by caspases.


Human autoimmune sera as molecular probes for the identification of an autoantigen kinase signaling pathway.

Kamachi M, Le TM, Kim SJ, Geiger ME, Anderson P, Utz PJ - J. Exp. Med. (2002)

Human SRPK1 and SRPK2 are differentially cleaved by recombinant caspases in vitro. SRPK1 and SRPK2 were synthesized by in vitro transcription/translation and incubated with the recombinant caspases indicated by number at the top of each figure. Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and detected by autoradiography. The relative migration of molecular size markers in kilodaltons is indicated on the left side of each panel. Lanes are numbered at the bottom of each panel.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2194102&req=5

fig3: Human SRPK1 and SRPK2 are differentially cleaved by recombinant caspases in vitro. SRPK1 and SRPK2 were synthesized by in vitro transcription/translation and incubated with the recombinant caspases indicated by number at the top of each figure. Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and detected by autoradiography. The relative migration of molecular size markers in kilodaltons is indicated on the left side of each panel. Lanes are numbered at the bottom of each panel.
Mentions: Previous studies have demonstrated that an SRPK (topoisomerase I) is cleaved by caspases-3 and -6 at unconventional sites during apoptosis (23). The results shown in Fig. 1 further suggested that another SRPK (SRPK1), and perhaps other SRPKs, may also undergo proteolysis during apoptosis. We tested this hypothesis by using a well-validated in vitro cleavage assay using recombinant caspases. In vitro–transcribed/translated SRPKs were incubated in caspase cleavage buffer in the presence of recombinant caspases or a control bacterial lysate, separated by SDS-PAGE and analyzed by autoradiography. As shown in Fig. 3, A and B , human SRPK1 and SRPK2 are differentially cleaved by recombinant caspase-8 and caspase-9, respectively, yielding signature ∼66-kD and 34-kD fragments (SRPK1) and 80-kD and 50-kD fragments (SRPK2), respectively. None of the other caspases tested had the ability to cleave SRPK1 and SRPK2, although caspase-1 appeared to partially cleave human SRPK1, generating fragments of identical size to those observed with caspase-8 (lane 2). Mouse SRPK1 and SRPK2, which are 92 and 58% conserved between mouse and man, respectively, were also differentially cleaved by caspase-8 and caspase-9, respectively, generating fragments of approximately the same molecular weight (unpublished data). In contrast, none of the Clk/Sty isoforms was cleaved by recombinant caspases (unpublished data). Taken together with the published data on topoisomerase I, these results demonstrate that at least 3 of the 8 known SRPKs are differentially cleaved by caspases.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, CA 94305, USA.

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.

Show MeSH
Related in: MedlinePlus