The critical features and the mechanism of inhibition of a kinase interaction motif-based peptide inhibitor of JNK
The critical features and the mechanism of inhibition of a kinase interaction motif-based peptide inhibitor of JNK
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DOI：
10.1074/jbc.m402181200
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发表时间：
2004-08-27
影响因子：
4.8
通讯作者：
Bogoyevitch, MA
中科院分区：
文献类型：
--
作者：
Barr, RK;Boehm, I;Bogoyevitch, MA
我们先前报道了一种基于JIP-1的c-Jun N-末端激酶（JNK）结合结构域的氨基酸143-153的小肽作为JNK活性的体外抑制剂。该肽（TI-JIP：RP-KRPTTLNLF）类似于激酶相互作用基序（KIM =（K/R）（2-3）X1-6（L/I）X（L/I）），其为存在于MAPK级联中的上游激活剂、下游底物、磷酸酶和支架蛋白所共有。在这项研究中，我们的特点是JNK抑制这种肽的机制，并进一步研究了这种肽的生物化学特性，导致有效的JNK抑制。我们还测试了各种基于KIM的肽抑制JNK活性的能力。发现TI-JIP相对于磷酸受体底物c-Jun（K-I = 0.39 +/- 0.08 μ M）是竞争性的，并且相对于ATP表现出混合（非竞争性）抑制。我们测试的Pro-5的所有七个取代都显著降低了JNK抑制，改变Pro-5到Leu-8的间距也是如此。当我们独立地测试Thr-6或Thr-7的8个取代时，每个位置中只有一个取代是良好耐受的。此外，基于来自其他蛋白质的KIM的肽是显著不如TI-JIP有效的JNK抑制剂，包括来自JNK相互作用物Sab的肽，其含有TI-JIP中存在的所有关键抑制残基。因此，尽管先前已鉴定TI-JIP中的Arg-4、Pro-5、Leu-8和Leu-10对于介导JNK抑制独立地至关重要，但我们发现它们在其他11聚体肽中的存在对于JNK抑制是不够的。因此，TI-JIP是一种独特的基于KIM的JNK活性抑制剂。
We previously reported that a small peptide based on amino acids 143-153 of the c-Jun N-terminal kinase (JNK)-binding domain of JIP-1 functioned as an in vitro inhibitor of JNK activity. This peptide (TI-JIP: RP-KRPTTLNLF) resembles the kinase-interaction motif (KIM = (K/R)(2-3)X1-6(L/I)X(L/I)), which is common to upstream activators, downstream substrates, phosphatases, and scaffold proteins present in MAPK cascades. In this study, we characterized the mechanism of JNK inhibition by this peptide and further investigated the biochemical features of this peptide resulting in potent JNK inhibition. We also tested various KIM-based peptides for their ability to inhibit JNK activity. TI-JIP was found to be competitive with respect to the phosphoacceptor substrate c-Jun (K-I = 0.39 +/- 0.08 muM), and exhibit mixed (non-competitive) inhibition with respect to ATP. All seven substitutions of Pro-5 we tested significantly reduced the JNK inhibition, as did altering the Pro-5 to Leu-8 spacing. When we independently tested eight substitutions of either Thr-6 or Thr-7, only one substitution in each position was well tolerated. Furthermore, peptides based on the KIMs from other proteins were significantly less potent JNK inhibitors than TI-JIP, including a peptide from the JNK interactor Sab that contained all critical inhibitory residues present in TI-JIP. Therefore, despite having previously identified Arg-4, Pro-5, Leu-8, and Leu-10 in TI-JIP as independently critical for mediating JNK inhibition, we find their presence in other 11-mer peptides is not sufficient for JNK inhibition. TI-JIP is therefore a unique KIM-based inhibitor of JNK activity.