Kamachi Makoto, Le Truc M, Kim Susan J, Geiger Meghan E, Anderson Paul, Utz Paul J
Department of Medicine, Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, CA 94305, USA.
J Exp Med. 2002 Nov 4;196(9):1213-25. doi: 10.1084/jem.20021167.
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.
利用人类自身免疫血清作为分子探针，我们先前描述了磷酸化丝氨酸/精氨酸剪接因子（SR剪接因子）与凋亡细胞中的U1小核核糖核蛋白（U1-snRNP）和U3小核仁核糖核蛋白（snoRNP）的关联。SR蛋白是高度保守的自身抗原，其活性受到至少八种不同的SR蛋白激酶（SRPK）对丝氨酸残基可逆磷酸化的严格调控，包括SRPK1、SRPK2以及硬皮病自身抗原拓扑异构酶I。在本报告中，我们证明在健康细胞和凋亡细胞中，已知的SRPK中只有一种，即SRPK1，与U1-snRNP自身抗原复合物相关联。SRPK1在凋亡早期被激活，随后在后期被半胱天冬酶介导的蛋白水解失活。在多种凋亡刺激后，SRPK在体内被切割，并且这种切割可被bcl-2和bcl-x(L)的过表达以及可溶性肽半胱天冬酶抑制剂所抑制。重组半胱天冬酶与体外翻译的SRPK一起孵育表明，SRPK1和SRPK2分别是半胱天冬酶-8和-9的体外底物。相比之下，拓扑异构酶I被下游半胱天冬酶（-3和-6）切割。由于这些SRPK中的每一种都处于半胱天冬酶级联反应的不同检查点，SRPK可能在调控凋亡、可变mRNA剪接、SR蛋白运输、RNA稳定性以及可能针对剪接因子的自身抗体产生的信号通路中发挥重要作用。
