Development of phosphatase inhibitor-1 peptides acting as indirect activators of phosphatase 1
Development of phosphatase inhibitor-1 peptides acting as indirect activators of phosphatase 1
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DOI：
10.1007/s00210-014-1065-2
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发表时间：
2015-03-01
影响因子：
3.6
通讯作者：
Eschenhagen, Thomas
中科院分区：
文献类型：
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作者：
Sotoud, Hannieh;Borgmeyer, Uwe;Eschenhagen, Thomas
磷酸酶抑制剂-1（I-1）在其蛋白激酶A (PKA)-磷酸化形式（I-1(P)）中抑制蛋白磷酸酶1型（PP1c）的催化亚基。因此，它放大了PKA信号，在心脏中，介导儿茶酚胺的有益（急性）和不良（慢性）效应。I-1基因缺失与儿茶酚胺毒性保护有关，使PP1c-I-1(P)复合物成为慢性心脏病的潜在治疗靶点。在这里，我们试图定义I-1和PP1c的可靶向相互作用位点，集中在I-1的n端结构域，包括PP1c结合基序(（9)KIQF(12)）以及poly-Arg拉伸。(9)KIQ（11）残基被类似氨基酸(RLN11)-R-9取代，导致IC50值加倍，(9)KIQF（12）的缺失阻止了I-1 pka依赖性磷酸化从而激活。在PKA磷酸化位点（Thr35）到Ala （R/A(30-33)）之前的Arg残基突变消除了I-1磷酸化及其与PP1c的结合和抑制。一系列合成肽（4-11个残基）表明，KIQF基序及其周围的锚定残基对干扰I-1(P)对PP1c的抑制作用至关重要，而4个Arg残基则不是。出乎意料的是，最有效的非肽（SPRKIQFTV）也以相似的亲和力拮抗了非条件PP1抑制剂-2的抑制作用。用聚精氨酸修饰的SPRKIQFTV （10 μ M）孵育新生大鼠心肌细胞，可以降低儿茶酚胺诱导的磷蛋白磷酸化，磷蛋白是一种众所周知的对PP1c敏感的PKA下游靶点。我们的数据重申了KIQF基序的重要性，并提供了一种工具来拮抗I-1对PP1c的抑制作用，即在体内激活PP1。
Phosphatase inhibitor-1 (I-1) inhibits the catalytic subunit of protein phosphatase type 1 (PP1c) in its protein kinase A (PKA)-phosphorylated form (I-1(P)). It thereby amplifies PKA signaling, which, in the heart, mediates both beneficial (acute) and adverse (chronic) effects of catecholamines. Genetic deletion of I-1 was associated with protection against catecholamine toxicity, making the PP1c-I-1(P) complex a potential therapeutic target for chronic heart disease. Here, we sought to define targetable interaction sites of I-1 and PP1c, concentrating on the N-terminal domain of I-1 which includes the PP1c binding motif ((9)KIQF(12)) as well as a poly-Arg stretch. Substitution of (9)KIQ(11) residues for analogous amino acids, (RLN11)-R-9, resulted in doubling of the IC50 values, deletion of (9)KIQF(12) prevented I-1 PKA-dependent phosphorylation and thus activation. Mutation of the Arg residues preceding the PKA phosphorylation site (Thr35) to Ala (R/A(30-33)) abolished I-1 phosphorylation and its binding to and inhibition of PP1c. A series of synthetic peptides (4-11 residues) indicated that the KIQF motif as well as the surrounding anchoring residues was essential for interfering with the inhibitory effect of I-1(P) on PP1c, whereas the four Arg residues were not. Unexpectedly, the most effective nonapeptide (SPRKIQFTV) also antagonized the inhibitory effect of the non-conditional PP1 inhibitor-2 with similar affinity. Incubation of neonatal rat cardiac myocytes with a poly-Arg-modified SPRKIQFTV (10 mu M) reduced catecholamine-induced phosphorylation of phospholamban, a well-known PKA downstream target sensitive to PP1c. Our data reiterate the importance of the KIQF motif and provide a tool for antagonizing I-1 inhibitory effects on PP1c, i.e., activating PP1 in vivo.