Akt and mitogen-activated protein kinase enhance C-type lectin-like receptor 2-mediated platelet activation by inhibition of glycogen synthase kinase 3α/β.
Akt and mitogen-activated protein kinase enhance C-type lectin-like receptor 2-mediated platelet activation by inhibition of glycogen synthase kinase 3α/β.
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DOI：
10.1111/jth.12954
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发表时间：
2015-06
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影响因子：
--
通讯作者：
Watson SP
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文献类型：
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作者：
Moroi AJ;Watson SP
C型凝集素样受体2（CLEC-2）和胶原受体糖蛋白（GP）VI通过Src和Syk酪氨酸激酶以及磷脂酶Cγ2激活血小板。然而，两个信号级联的初始事件是不同的，并且Syk激活下游蛋白质的作用存在定量差异。Akt和丝裂原活化蛋白激酶（MAPK）的活化已被证明可增强GPVI对血小板的活化，但它们在CLEC-2信号传导中的作用尚不清楚。我们试图研究Akt和MAPK通路在CLEC-2激活血小板中的作用。CLEC-2激动剂rhodocytin刺激Akt和p38以及细胞外信号相关激酶（ERK）MAPK的磷酸化，但相对于Syk有延迟。在腺苷三磷酸双磷酸酶和吲哚美辛的组合存在下，这些蛋白的磷酸化被显著抑制，这与报道的ADP和血栓烷A2在CLEC-2信号传导中的反馈作用一致。Akt的磷酸化和ERK的磷酸化分别被磷酸肌醇3-激酶（PI 3 K）抑制剂渥曼青霉素和蛋白激酶C（PKC）抑制剂Ro 31 - 8220阻断，而Syk磷酸化没有改变。另一方面，两种抑制剂均降低Akt底物糖原合成酶激酶3α/β（GSK 3 α/β）的磷酸化。GSK 3 α/β的磷酸化也被Akt抑制剂MK2206阻断，并且在后期而不是早期被MEK抑制剂PD 0325901降低。MK 2206和PD 0325901抑制了对低浓度红细胞素的反应的聚集和分泌，这可通过GSK 3 α/β抑制剂恢复。这些结果表明，CLEC-2调节PI 3 K和PKC下游的Akt和MAPK，导致GSK 3 α/β磷酸化和抑制，并增强血小板聚集和分泌。
The C‐type lectin‐like receptor 2 (CLEC‐2) and the collagen receptor glycoprotein (GP)VI activate platelets through Src and Syk tyrosine kinases, and phospholipase Cγ2. The initial events in the two signaling cascades, however, are distinct, and there are quantitative differences in the roles of proteins downstream of Syk activation. The activation of Akt and mitogen‐activated protein kinases (MAPKs) has been shown to enhance platelet activation by GPVI, but their role in CLEC‐2 signaling is not known. We sought to investigate the role of the Akt and MAPK pathways in platelet activation by CLEC‐2. The CLEC‐2 agonist rhodocytin stimulated phosphorylation of Akt and p38 and extracellular signal‐related kinase (ERK) MAPKs, but with a delay relative to Syk. Phosphorylation of these proteins was markedly inhibited in the combined presence of apyrase and indomethacin, consistent with the reported feedback action of ADP and thromboxane A2 in CLEC‐2 signaling. Phosphorylation of Akt and phosphorylation of ERK were blocked by the phosphoinositide 3‐kinase (PI3K) inhibitor wortmannin and the protein kinase C (PKC) inhibitor Ro31‐8220, respectively, whereas Syk phosphorylation was not altered. On the other hand, both inhibitors reduced phosphorylation of the Akt substrate glycogen synthase kinase 3α/β (GSK3α/β). Phosphorylation of GSK3α/β was also blocked by the Akt inhibitor MK2206, and reduced at late, but not early, times by the MEK inhibitor PD0325901. MK2206 and PD0325901 inhibited aggregation and secretion in response to a low concentration of rhodocytin, which was restored by GSK3α/β inhibitors. These results demonstrate that CLEC‐2 regulates Akt and MAPK downstream of PI3K and PKC, leading to phosphorylation and inhibition of GSK3α/β, and enhanced platelet aggregation and secretion.