PDK1 selectively phosphorylates Thr(308) on Akt and contributes to human platelet functional responses.
PDK1 selectively phosphorylates Thr(308) on Akt and contributes to human platelet functional responses.
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DOI：
10.1160/th13-06-0484
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发表时间：
2014-03-03
影响因子：
6.7
通讯作者：
Kunapuli SP
中科院分区：
文献类型：
--
作者：
Dangelmaier C;Manne BK;Liverani E;Jin J;Bray P;Kunapuli SP
3-磷酸肌醇依赖性蛋白激酶1（PDK 1）是蛋白A、G和C（AGC）家族的成员，是一种Ser/Thr蛋白激酶，其可以磷酸化并激活AGC家族的其他蛋白激酶，包括Thr 308处的Akt，所有这些蛋白激酶在介导细胞应答中起重要作用。尚未在人血小板中研究PDK 1的功能作用或Thr 308上Akt磷酸化对其活性的重要性。在这项研究中，我们测试了两种PDK 1的药理学抑制剂BX 795和BX 912，以评估Thr 308磷酸化对Akt的作用。PAR 4诱导的Akt在Thr 308上的磷酸化被BX 795抑制，而不影响Akt在Ser 473上的磷酸化。Akt上Thr 308磷酸化的缺乏也导致对Akt的两种下游底物（即GSK 3 β和PRAS 40）的PAR 4诱导的磷酸化的抑制。如果Akt上的Thr 308不被磷酸化，则Akt的体外激酶活性被完全消除。BX 795抑制2-MeSADP诱导的或胶原诱导的聚集、ATP分泌和血栓素生成。在BX 795的存在下，2-MeSADP诱导的初级聚集也被抑制。PDK 1抑制还导致血块收缩减少，表明其在由外向内信号传导中的作用。这些结果表明，PDK 1选择性磷酸化Akt上的Thr 308，从而调节其活性，并在血小板生理反应中发挥积极的调节作用。
3-phosphoinositide-dependent protein kinase 1 (PDK1), a member of the protein A,G and C (AGC) family of proteins, is a Ser/Thr protein kinase that can phosphorylate and activate other protein kinases from the AGC family, including Akt at Thr308, all of which play important roles in mediating cellular responses. The functional role of PDK1 or the importance of phosphorylation of Akt on Thr308 for its activity has not been investigated in human platelets. In this study, we tested two pharmacological inhibitors of PDK1, BX795 and BX912, to assess the role of Thr308 phosphorylation on Akt. PAR4-induced phosphorylation of Akt onThr308 was inhibited by BX795 without affecting phosphorylation of Akt on Ser473. The lack of Thr308 phosphorylation on Akt also led to the inhibition of PAR4-induced phosphorylation of two downstream substrates of Akt, viz. GSK3β and PRAS40. In vitro kinase activity of Akt was completely abolished if Thr308 on Akt was not phosphorylated. BX795 caused inhibition of 2-MeSADP-induced or collagen-induced aggregation, ATP secretion and thromboxane generation. Primary aggregation induced by 2-MeSADP was also inhibited in the presence of BX795. PDK1 inhibition also resulted in reduced clot retraction indicating its role in outside-in signalling. These results demonstrate that PDK1 selectively phosphorylates Thr308 on Akt thereby regulating its activity and plays a positive regulatory role in platelet physiological responses.