Characterization of a ceramide kinase-like protein
Characterization of a ceramide kinase-like protein
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DOI：
10.1016/j.bbalip.2004.11.012
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发表时间：
2005-02-21
影响因子：
4.8
通讯作者：
Billich, A
中科院分区：
文献类型：
--
作者：
Bornancin, FR;Mechtcheriakova, D;Billich, A
神经酰胺是决定细胞命运的关键因素，神经酰胺酶(Cerk)将其转化为神经酰胺-1-磷酸，是调节细胞凋亡和炎症过程的重要手段。我们鉴定了一个新的神经酰胺激酶同源物，命名为CERK样蛋白(CERKL)，并将其与已知的CERK进行了比较。实时定量-聚合酶链式反应分析揭示了CERKL基因表达的限制性模式。令人惊讶的是，CERKL在体外不能将多种神经酰胺(已知的CEK底物)磷酸化。用~(32)P(I)脉冲标记瞬时表达CERKL的COS-1细胞，或与NBD-C6-神经酰胺孵育，未检测到神经酰胺-1-磷酸。在COS-1细胞中重组表达后，CERKL以磷酸化蛋白的形式部分恢复到可溶性部分。活细胞成像显示GFP标记的CERKL定位于许多细胞室，包括与核仁的特异性结合。CERKL的两个剪接变异体没有定位到核仁，一个CERKL变异体也没有定位到核仁上，在推测的ATP结合位点上有一个点突变。我们还研究了一个自然发生的CERKL突变(R257X)，最近与视网膜色素变性的病理有关。它聚集在细胞核内，但与核仁无关。用钙离子载体A23187处理后，CERKL从核仁中清除，但对R257X CERKL突变体没有影响。总之，尽管CERKL的激酶活性仍有待证实，但这些发现表明CERKL与其核仁定位之间存在功能联系。此外，我们认为携带CERKL R257X突变的患者中视网膜色素变性的原因可能是截短的CERKL蛋白在细胞核内积聚。(C)2004年，爱思唯尔出版。
Ceramide is a key player governing cell fate, and its conversion to ceramide-1-phosphate by ceramide kinase (CERK) is emerging as an important mean to regulate apoptosis and inflammatory processes. We identified a new ceramide kinase homolog, designated CERK-like protein (CERKL) and we compared it to the known CERK. Real time-PCR analysis of human tissues revealed a restricted pattern of expression for CERKL mRNA. Surprisingly, various ceramides, known substrates for CERK, were not phosphorylated by CERKL in vitro. Upon (32)p(i)-pulse labeling of COS-1 cells transiently expressing CERKL, or incubation with NBD-C6-ceramide, ceramide-1-phosphate was not detected. After recombinant expression in COS-1 cells, CERKL was partially recovered in the soluble fraction, as a phosphorylated protein. Live cell imaging indicated localization of GFP-tagged CERKL to many cell compartments, including specific association with nucleoli. Two splice variants of CERKL did not localize to nucleoli nor did a CERKL variant with a point mutation in the putative ATP binding site. We also studied a naturally occurring CERKL mutant (R257X), recently linked to the pathology of retinitis pigmentosa. It accumulated in the nucleus but was not associated with nucleoli. Treatment with the calcium ionophore A23187 led to clearing of CERKL from nucleoli, but had no effect on the R257X CERKL mutant. Collectively, although kinase activity of CERKL remains to be proven, these findings suggest a functional link between CERKL and its nucleolar localization. Furthermore, we propose that the cause for retinitis pigmentosa in patients bearing the CERKL R257X mutation might be the accumulation of a truncated CERKL protein in the nucleus. (C) 2004 Published by Elsevier B.V.