The N Terminus of Cbl-c Regulates Ubiquitin Ligase Activity by Modulating Affinity for the Ubiquitin-conjugating Enzyme
The N Terminus of Cbl-c Regulates Ubiquitin Ligase Activity by Modulating Affinity for the Ubiquitin-conjugating Enzyme
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DOI：
10.1074/jbc.m109.091157
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发表时间：
2010-07-30
影响因子：
4.8
通讯作者：
Lipkowitz, Stanley
中科院分区：
文献类型：
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作者：
Ryan, Philip E.;Sivadasan-Nair, Nina;Lipkowitz, Stanley
Cbl 蛋白是泛素连接酶 (E3)，在调节酪氨酸激酶信号传导中发挥重要作用。哺乳动物家族共有三个成员：Cbl、Cbl-b 和 Cbl-c。所有这些都具有高度保守的 N 端酪氨酸激酶结合结构域、催化环指结构域和 C 端富含脯氨酸的结构域，可介导与含有 Src 同源性 3 (SH3) 的蛋白质的相互作用。尽管 Cbl 和 Cbl-b 都已被广泛研究，但对 Cbl-c 却知之甚少。已发表的报告表明，Cbl和Cbl-b的N末端对其各自的E3活性具有抑制作用。然而，这种抑制的机制仍不清楚。在本研究中，我们证明 Cbl-c 的 N 末端与 Cbl 和 Cbl-b 的 N 末端一样，抑制 Cbl-c 的 E3 活性。此外，我们将负责抑制的区域映射到 EF-hand 和 SH2 结构域。 Cbl-c 连接区中的关键酪氨酸 (Tyr-341) 通过 Src 或该酪氨酸的磷酸模拟突变 (Y341E) 进行磷酸化，足以增加 Cbl-c 的 E3 活性。我们还首次证明 Tyr-341 磷酸化或 Y341E 突变会导致对泛素结合酶 (E2) UbcH5b 的亲和力降低。 Y341E 突变体 Cbl-c 对 UbcH5b 的亲和力降低导致结合的 UbcH5b 更快速的周转，同时 E3 活性增加。这些数据表明，Cbl-c 的 N 末端有助于与 E2 的结合，并且 Tyr-341 的磷酸化导致 Cbl-c 的亲和力降低和 E3 活性增加。
Cbl proteins are ubiquitin ligases (E3s) that play a significant role in regulating tyrosine kinase signaling. There are three mammalian family members: Cbl, Cbl-b, and Cbl-c. All have a highly conserved N-terminal tyrosine kinase binding domain, a catalytic RING finger domain, and a C-terminal proline-rich domain that mediates interactions with Src homology 3 (SH3) containing proteins. Although both Cbl and Cbl-b have been studied widely, little is known about Cbl-c. Published reports have demonstrated that the N terminus of Cbl and Cbl-b have an inhibitory effect on their respective E3 activity. However, the mechanism for this inhibition is still unknown. In this study we demonstrate that the N terminus of Cbl-c, like that of Cbl and Cbl-b, inhibits the E3 activity of Cbl-c. Furthermore, we map the region responsible for the inhibition to the EF-hand and SH2 domains. Phosphorylation of a critical tyrosine (Tyr-341) in the linker region of Cbl-c by Src or a phosphomimetic mutation of this tyrosine (Y341E) is sufficient to increase the E3 activity of Cbl-c. We also demonstrate for the first time that phosphorylation of Tyr-341 or the Y341E mutation leads to a decrease in affinity for the ubiquitin-conjugating enzyme (E2), UbcH5b. The decreased affinity of the Y341E mutant Cbl-c for UbcH5b results in a more rapid turnover of bound UbcH5b coincident with the increased E3 activity. These data suggest that the N terminus of Cbl-c contributes to the binding to the E2 and that phosphorylation of Tyr-341 leads to a decrease in affinity and an increase in the E3 activity of Cbl-c.