Phosphorylation of the alternative mRNA splicing factor 45 (SPF45) by Clk1 regulates its splice site utilization, cell migration and invasion.
Phosphorylation of the alternative mRNA splicing factor 45 (SPF45) by Clk1 regulates its splice site utilization, cell migration and invasion.
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DOI：
10.1093/nar/gkt170
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发表时间：
2013-05
影响因子：
14.9
通讯作者：
Eblen ST
中科院分区：
文献类型：
--
作者：
Liu Y;Conaway L;Rutherford Bethard J;Al-Ayoubi AM;Thompson Bradley A;Zheng H;Weed SA;Eblen ST
选择性mRNA剪接是调节蛋白异构体表达的一种机制，受选择性剪接因子的调控。选择性剪接因子45 （alternative splicing factor 45, SPF45）在肿瘤中过度表达，尽管目前对其生物学效应知之甚少，也很少发现剪接靶点。我们之前的研究表明，细胞外调节激酶2 （ERK2）磷酸化的SPF45调节细胞增殖和对纤维连接蛋白的粘附。在这项工作中，我们发现cdc2样激酶1 （Clk1）在8个丝氨酸残基上磷酸化SPF45。Clk1表达增强，而Clk1抑制减弱，spf45诱导Fas mRNA外显子6被排除。对SPF45上Clk1磷酸化位点的突变分析显示，剪接有正调控和负调控，净效应是抑制SPF45诱导的外显子6排除，与Fas mRNA结合减少相关。然而，Clk1增强了SPF45蛋白的表达，而不是mRNA的表达，而抑制Clk1通过蛋白酶体依赖途径增加了SPF45的降解。过表达SPF45或磷酸化模拟突变体，而非磷酸化抑制突变体，刺激卵巢癌细胞迁移和侵袭，与纤维连接蛋白表达增加、ERK激活和全长接触蛋白剪接和磷酸化增强相关。我们的研究结果首次证明，SPF45过表达增强细胞迁移和侵袭，依赖于Clk1的生化调节。
Alternative mRNA splicing is a mechanism to regulate protein isoform expression and is regulated by alternative splicing factors. The alternative splicing factor 45 (SPF45) is overexpressed in cancer, although few biological effects of SPF45 are known, and few splicing targets have been identified. We previously showed that Extracellular Regulated Kinase 2 (ERK2) phosphorylation of SPF45 regulates cell proliferation and adhesion to fibronectin. In this work, we show that Cdc2-like kinase 1 (Clk1) phosphorylates SPF45 on eight serine residues. Clk1 expression enhanced, whereas Clk1 inhibition reduced, SPF45-induced exon 6 exclusion from Fas mRNA. Mutational analysis of the Clk1 phosphorylation sites on SPF45 showed both positive and negative regulation of splicing, with a net effect of inhibiting SPF45-induced exon 6 exclusion, correlating with reduced Fas mRNA binding. However, Clk1 enhanced SPF45 protein expression, but not mRNA expression, whereas inhibition of Clk1 increased SPF45 degradation through a proteasome-dependent pathway. Overexpression of SPF45 or a phospho-mimetic mutant, but not a phospho-inhibitory mutant, stimulated ovarian cancer cell migration and invasion, correlating with increased fibronectin expression, ERK activation and enhanced splicing and phosphorylation of full-length cortactin. Our results demonstrate for the first time that SPF45 overexpression enhances cell migration and invasion, dependent on biochemical regulation by Clk1.
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