Cell Biology and Gene Expression Unit, Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, Maryland, United States of America.
PLoS One. 2010 Jan 15;5(1):e8730. doi: 10.1371/journal.pone.0008730.
Mutations in the gene encoding Leucine-rich repeat kinase 2 (LRRK2) are the most common cause of inherited Parkinson's disease (PD). LRRK2 is a multi-domain protein kinase containing a central catalytic core and a number of protein-protein interaction domains. An important step forward in the understanding of both the biology and the pathology of LRRK2 would be achieved by identification of its authentic physiological substrates. In the present study we examined phosphorylation of 4E-BP (eukaryotic initiation factor 4E (eIF4E)-binding protein), a recently proposed substrate for LRRKs. We found that LRRK2 is capable of phosphorylating 4E-BP in vitro. The PD related LRRK2-G2019S mutant was approximately 2 fold more active than wild type protein. However, LRRK2 autophosphorylation was stronger than 4E-BP phosphorylation under conditions of molar excess of 4E-BP to LRRK2. We also tested three other kinases (STK3, MAPK14/p38alpha and DAPK2) and found that MAPK14/p38alpha could efficiently phosphorylate 4E-BP at the same site as LRRK2 in vitro. Finally, we did not see changes in 4E-BP phosphorylation levels using inducible expression of LRRK2 in HEK cell lines. We also found that MAPK14/p38alpha phosphorylates 4E-BP in transient overexpression experiments whereas LRRK2 did not. We suggest that increased 4E-BP phosphorylation reported in some systems may be related to p38-mediated cell stress rather than direct LRRK2 activity. Overall, our results suggest that 4E-BP is a relatively poor direct substrate for LRRK2.
LRRK2 基因编码的突变是遗传性帕金森病 (PD) 的最常见原因。LRRK2 是一种含有中央催化核心和多个蛋白-蛋白相互作用结构域的多结构域蛋白激酶。鉴定其真实的生理底物，将是在理解 LRRK2 的生物学和病理学方面的重要进展。在本研究中，我们检测了 4E-BP（真核起始因子 4E (eIF4E)-结合蛋白）的磷酸化，这是最近提出的 LRRK2 的底物。我们发现 LRRK2 能够在体外磷酸化 4E-BP。与野生型蛋白相比，PD 相关的 LRRK2-G2019S 突变体的活性约增加了 2 倍。然而，在 4E-BP 与 LRRK2 的摩尔比过量的情况下，LRRK2 的自身磷酸化比 4E-BP 的磷酸化要强。我们还测试了另外三种激酶 (STK3、MAPK14/p38alpha 和 DAPK2)，并发现 MAPK14/p38alpha 可以在体外有效地在与 LRRK2 相同的位点磷酸化 4E-BP。最后，我们在 HEK 细胞系中用诱导表达 LRRK2 的方法，没有观察到 4E-BP 磷酸化水平的变化。我们还发现，在瞬时过表达实验中，MAPK14/p38alpha 磷酸化 4E-BP，而 LRRK2 则没有。我们认为，一些系统中报道的 4E-BP 磷酸化增加可能与 p38 介导的细胞应激有关，而不是直接与 LRRK2 的活性有关。总的来说，我们的结果表明 4E-BP 是 LRRK2 的相对较差的直接底物。
