促红细胞生成素产生肝细胞受体A2(EphA2)SAM结构域对激酶结构域的脂质体结合能力与激酶活性的调控研究

促红细胞生成素产生肝细胞受体(Eph receptor) 是受体酪氨酸激酶(RTK)家族中最大的亚家族,其介导的双向信号传导对细胞的形态、黏附、运动、增殖、生存及分化都有重要的调控作用。EphA2是Eph受体家族中一个被广泛研究的重要亚型,在白内障和乳腺癌等病理发生过程中发挥了重要作用。既往研究发现:EphA2受体的激酶结构域可结合细胞膜,其激酶活性受磷脂膜的调控,但是相邻的SAM结构域对激酶结构域与脂膜的相互作用以及激酶活性的影响尚不清楚。在此项研究中,通过与磷酸酶PTP1B1-301活性片段共表达的方式,表达、纯化了EphA2受体的胞内段激酶-SAM串联结构域,通过比较胞内段激酶-SAM串联结构域与单独激酶结构域的脂质体结合能力,以及测定对应的激酶活性,发现:EphA2受体胞内段的SAM结构域使其激酶结构域与脂质体(4 mg/mL)的结合能力增强约6倍(P＜0.001);磷酸化后的EphA2胞内段激酶-SAM串联结构域结合脂质体(4 mg/mL)的能力比非磷酸化的胞内段激酶-SAM串联结构域提高2.5倍(P＜0.05);而结合脂质体后,激酶结构域的激酶活性也被进一步提高,从而形成正反馈。综上所述,本研究的发现提示:EphA2胞内段的酪氨酸激酶结构域与相邻的SAM结构域可形成一个完整的结构功能单位,其激酶活性和脂质体结合能力与单独的激酶结构域相比都形成了明显的差异,我们的这一发现对进一步理解Eph受体家族其他亚型的激酶结构域的活性调控提供了参考与思路。     

泛素-蛋白酶体降解途径在细胞周期调控中的作用

细胞周期的进程由一系列细胞周期蛋白依赖性激酶(CDK)和CDK活性调节因子驱动。泛素-蛋白酶体对细胞周期调节因子的降解是细胞调控分裂进程的重要手段。CDK活性抑制因子的降解是细胞分裂所必需的,而细胞周期正调控因子的降解则对维持细胞稳态至关重要。本从参与调控的2类泛素连接酶SCF复合物、APC／C复合物的结构和功能的角度阐述了泛素-蛋白酶体降解途径在整个细胞周期调控中的作用和意义。     

泛素结合酶UBE2Q2参与HUWE1对肾小管间质纤维化的抑制作用

泛素-蛋白酶体系统在蛋白质降解时发挥着重要的作用.泛素化过程需要E1泛素激活酶、E2泛素结合酶、E3泛素连接酶协同完成.本研究组前期研究证明E3泛素连接酶HUWE1(HECT,UBA and WWE domain containing 1)可降解表皮生长因子受体(epidermal growth factor rece...     

快速高效检测植物体内蛋白泛素化修饰研究方法

UPS参与植物中绝大多数的信号转导通路。其中, 一些激素的受体本身就是E3泛素连接酶, 如茉莉酸(JA)受体COI1和生长素(auxin)受体TIR1都是F-box蛋白, 它们通过特异性介导相应转录抑制子的泛素化降解来传递激素信号, 但对于整个UPS体系而言, 由于技术的限制, 迄今为止仅见少量泛素连接酶与特异性底物间生化机制的报道。用大肠杆菌(Escherichia coli)表达蛋白实施泛素连接酶泛素化修饰底物的体外实验是验证泛素连接酶/底物对的常用方法, 但由于体外实验缺乏某些蛋白必需的转录后修饰, 导致实验结果有时存在假阴性。利用农杆菌注射烟草(Nicotiana benthamiana)瞬时表达蛋白的方法, 建立高效的植物体内检测蛋白泛素化系统, 可以快速检测蛋白泛素化, 包括检测泛素连接酶和底物的特异性相互作用、底物蛋白的自身泛素化、泛素连接酶对底物降解的促进作用、26S蛋白酶体抑制剂MG132对底物降解的抑制作用以及用植物内源表达蛋白进行体外泛素化反应。     

泛素化和磷酸化协同作用调控蛋白质降解

在真核细胞中,泛素化和磷酸化是2种常见的蛋白质修饰方式。泛素在蛋白酶体降解途径中发挥重要的靶向作用,细胞外信号严格调控着目的蛋白的泛素化。在很多情况下,这种调控依赖于蛋白质的磷酸化。由磷酸化影响的调控步骤可能与E3泛素连接酶对底物的识别有关,也可能与实际的交联反应有关。这种调控是通过对底物或E3连接酶本身的磷酸化实现的。     

泛素/26S蛋白酶体途径与植物的生长发育

泛素/26S蛋白酶体途径在植物蛋白降解系统中起重要作用,泛素分子主要通过泛素活化酶(E1)、泛素结合酶(E2)和泛素连接酶(E3)将靶蛋白泛素化,泛素化的蛋白最后被26S蛋白酶体识别和降解。泛素蛋白酶体途径参与植物体内的多种生理过程,如花和胚的发育、光形态建成、植物生长物质等几乎所有的生长发育过程,本文主要对泛素/26S蛋白酶体途径及其在植物生长发育过程中的精确调控作用进行综述。     

快速高效检测植物体内蛋白泛素化修饰研究方法

UPS参与植物中绝大多数的信号转导通路。其中, 一些激素的受体本身就是E3泛素连接酶, 如茉莉酸(JA)受体COI1和生长素(auxin)受体TIR1都是F-box蛋白, 它们通过特异性介导相应转录抑制子的泛素化降解来传递激素信号, 但对于整个UPS体系而言, 由于技术的限制, 迄今为止仅见少量泛素连接酶与特异性底物间生化机制的报道。用大肠杆菌(Escherichia coli)表达蛋白实施泛素连接酶泛素化修饰底物的体外实验是验证泛素连接酶/底物对的常用方法, 但由于体外实验缺乏某些蛋白必需的转录后修饰, 导致实验结果有时存在假阴性。利用农杆菌注射烟草(Nicotiana benthamiana)瞬时表达蛋白的方法, 建立高效的植物体内检测蛋白泛素化系统, 可以快速检测蛋白泛素化, 包括检测泛素连接酶和底物的特异性相互作用、底物蛋白的自身泛素化、泛素连接酶对底物降解的促进作用、26S蛋白酶体抑制剂MG132对底物降解的抑制作用以及用植物内源表达蛋白进行体外泛素化反应。     

泛素/26S蛋白酶体途径及其在植物生长发育中的功能

泛素/26S蛋白酶体途径是一种蛋白高效降解途径,主要负责真核细胞内蛋白的选择性降解.泛素分子主要通过泛素活化酶E1、泛素结合酶E2和泛素-蛋白连接酶E3将靶蛋白泛素化,泛素化的蛋白最后被26S蛋白酶体识别和降解.本文介绍了泛素/26S蛋白体介导的特异性蛋白质降解途经,并对其在植物激素信号、光形态建成、植物衰老、自交不亲和反应、细胞周期调控、花的发育、生物钟节律和非生物胁迫响应中的功能最新研究进展进行了综述.     

泛素蛋白酶体途径及其对植物生长发育的调控

泛素蛋白酶体途径主要由泛素活化酶、泛素结合酶、泛素蛋白连接酶和26S蛋白酶体组成。泛素活化酶首先激活泛素分子,然后把泛素转移到泛素结合酶上。泛素结合酶结合泛素蛋白连接酶并把泛素转移到底物蛋白上使底物泛素化,或把泛素转移到泛素蛋白连接酶再使底物泛素化。泛素化的蛋白通常通过26S蛋白酶体进行降解。初步的研究结果表明,植物生长发育的很多方面受泛素蛋白酶体介导的蛋白降解途径的调控。     

Negative regulation of EphA2 receptor by Cbl

The c-Cbl proto-oncogene product Cbl has emerged as a negative regulator of receptor and non-receptor tyrosine kinases, a function dependent on its recently identified ubiquitin ligase activity. Here, we report that EphA2, a member of Eph receptor tyrosine kinases is negatively regulated by Cbl. The negative regulation of EphA2 mediated by Cbl is dependent on the activity of EphA2, as the kinase inactive mutant of EphA2 cannot be regulated by Cbl. Moreover, a point mutation (G306E-Cbl) in TKB region of Cbl that has been reported to abolish Cbl binding to RTKs and non-receptor tyrosine kinases impaired the binding to active EphA2. The dominant negative mutant 70Z-Cbl, which has a 17-amino acids deletion in the N-boundary of the RING finger domain, defuncted negative regulatory function of Cbl to EphA2. These results demonstrate that the TKB domain and RING finger domain of Cbl are essential for this negative regulation.     

Ephrin-A1 induces c-Cbl phosphorylation and EphA receptor down-regulation in T cells

Eph receptor tyrosine kinases are expressed by T lineage cells, and stimulation with their ligands, the ephrins, has recently been shown to modulate T cell behavior. We show that ephrin-A1 stimulation of Jurkat T cells induces tyrosine phosphorylation of EphA3 receptors and cytoplasmic proteins, including the c-cbl proto-oncogene. Cbl phosphorylation was also observed in peripheral blood T cells. In contrast, stimulation of Jurkat cells with the EphB receptor ligand ephrin-B1 does not cause Cbl phosphorylation. EphA activation also induced Cbl association with Crk-L and Crk-II adapters, but not the related Grb2 protein. Induction of Cbl phosphorylation upon EphA activation appeared to be dependent upon Src family kinase activity, as Cbl phosphorylation was selectively abrogated by the Src family inhibitor 4-amino-5(4-chlorophenyl-7-(tert-butyl)pyrazolo[3,4-d]pyrimidine, while EphA phosphorylation was unimpaired. Ephrin-A1 stimulation of Jurkat cells was also found to cause down-regulation of endogenous EphA3 receptors from the cell surface and their degradation. In accordance with the role of Cbl as a negative regulator of receptor tyrosine kinases, overexpression of wild-type Cbl, but not its 70-Z mutant, was found to down-regulate EphA receptor expression. Receptor down-regulation could also be inhibited by blockage of Src family kinase activity. Our findings show that EphA receptors can actively signal in T cells, and that Cbl performs multiple roles in this signaling pathway, functioning to transduce signals from the receptors as well as regulating activated EphA receptor expression.     

WW domain HECT E3s target Cbl RING finger E3s for proteasomal degradation

Cbl proteins have RING finger-dependent ubiquitin ligase (E3) activity that is essential for down-regulation of tyrosine kinases. Here we establish that two WW domain HECT E3s, Nedd4 and Itch, bind Cbl proteins and target them for proteasomal degradation. This is dependent on the E3 activity of the HECT E3s but not on that of Cbl. Consistent with these observations, in cells expressing the epidermal growth factor receptor, Nedd4 reverses Cbl-b effects on receptor down-regulation, ubiquitylation, and proximal events in signaling. Cbl-b also targets active Src for degradation in cells, and Nedd4 similarly reverses Cbl-mediated Src degradation. These findings establish that RING finger E3s can be substrates, not only for autoubiquitylation but also for ubiquitylation by HECT E3s and suggest an additional level of regulation for Cbl substrates including protein-tyrosine kinases.     

The non-receptor tyrosine kinase Syk is a target of Cbl-mediated ubiquitylation upon B-cell receptor stimulation.

The negative regulator Cbl functions as a ubiquitin ligase towards activated receptor tyrosine kinases and facilitates their transport to lysosomes. Whether Cbl ubiquitin ligase activity mediates its negative regulatory effects on cytoplasmic tyrosine kinases of the Syk/ZAP-70 family has not been addressed, nor is it known whether these kinases are regulated via ubiquitylation during lymphocyte B-cell receptor engagement. Here we show that B-cell receptor stimulation in Ramos cells induces the ubiquitylation of Syk tyrosine kinase which is inhibited by a dominant-negative mutant of Cbl. Intact tyrosine kinase-binding and RING finger domains of Cbl were found to be essential for Syk ubiquitylation in 293T cells and for in vitro Syk ubiquitylation. These same domains were also essential for Cbl-mediated negative regulation of Syk as measured using an NFAT-luciferase reporter in a lymphoid cell. Association with Cbl did not alter the kinase activity of Syk. Altogether, our results support an essential role for Cbl ubiquitin ligase activity in the negative regulation of Syk, and establish that ubiquitylation provides a mechanism of Cbl-mediated negative regulation of cytoplasmic targets.     

Binding of Cbl to a phospholipase Cgamma1-docking site on platelet-derived growth factor receptor beta provides a dual mechanism of negative regulation

Ubiquitin conjugation to receptor tyrosine kinases is a critical biochemical step in attenuating their signaling through lysosomal degradation. Our previous studies have established Cbl as an E3 ubiquitin ligase for ubiquitinylation and degradation of platelet-derived growth factor receptor (PDGFR) alpha and PDGFRbeta. However, the role of endogenous Cbl in PDGFR regulation and the molecular mechanisms of this regulation remain unclear. Here, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and degradation of PDGFRbeta; this involves the Cbl TKB domain binding to PDGFRbeta phosphotyrosine 1021, a known phospholipase C (PLC) gamma1 SH2 domain-binding site. Lack of Cbl or ablation of the Cbl-binding site on PDGFRbeta impedes receptor sorting to the lysosome. Cbl-deficient cells also show more PDGF-induced PLCgamma1 association with PDGFRbeta and enhanced PLC-mediated cell migration. Thus, Cbl-dependent negative regulation of PDGFRbeta involves a dual mechanism that concurrently promotes ubiquitin-dependent lysosomal sorting of the receptor and competitively reduces the recruitment of a positive mediator of receptor signaling.     

Met kinase-dependent loss of the E3 ligase Cbl in gastric cancer

Strict regulation of signaling by receptor tyrosine kinases (RTKs) is essential for normal biological processes, and disruption of this regulation can lead to tumor initiation and progression. Signal duration by the Met RTK is mediated in part by the E3 ligase Cbl. Cbl is recruited to Met upon kinase activation and promotes ubiquitination, trafficking, and degradation of the receptor. The Met RTK has been demonstrated to play a role in various types of cancer. Here, we show that Met-dependent loss of Cbl protein in MET-amplified gastric cancer cell lines represents another mechanism contributing to signal dysregulation. Loss of Cbl protein is dependent on Met kinase activity and is partially rescued with a proteasome inhibitor, lactacystin. Moreover, Cbl loss not only uncouples Met from Cbl-mediated negative regulation but also releases other Cbl targets, such as the EGF receptor, from Cbl-mediated signal attenuation. Thus, Met-dependent Cbl loss may also promote cross-talk through indirect enhancement of EGF receptor signaling.     

Cell Regulation by Phosphotyrosine-Targeted Ubiquitin Ligases

Three classes of E3 ubiquitin ligases, members of the Cbl, Hakai, and SOCS-Cul5-RING ligase families, stimulate the ubiquitination of phosphotyrosine-containing proteins, including receptor and nonreceptor tyrosine kinases and their phosphorylated substrates. Because ubiquitination frequently routes proteins for degradation by the lysosome or proteasome, these E3 ligases are able to potently inhibit tyrosine kinase signaling. Their loss or mutational inactivation can contribute to cancer, autoimmunity, or endocrine disorders, such as diabetes. However, these ligases also have biological functions that are independent of their ubiquitination activity. Here we review relevant literature and then focus on more-recent developments in understanding the structures, substrates, and pathways through which the phosphotyrosine-specific ubiquitin ligases regulate diverse aspects of cell biology.     

Receptor Tyrosine Kinase Ubiquitylation Involves the Dynamic Regulation of Cbl-Spry2 by Intersectin 1 and the Shp2 Tyrosine Phosphatase

Ubiquitylation of receptor tyrosine kinases (RTKs) regulates their trafficking and lysosomal degradation. The multidomain scaffolding protein intersectin 1 (ITSN1) is an important regulator of this process. ITSN1 stimulates ubiquitylation of the epidermal growth factor receptor (EGFR) through enhancing the activity of the Cbl E3 ubiquitin ligase. However, the precise mechanism through which ITSN1 enhances Cbl activity is unclear. Here, we demonstrate that ITSN1 interacts with and recruits the Shp2 tyrosine phosphatase to Spry2 to enhance its dephosphorylation, thereby disrupting the inhibitory effect of Spry2 on Cbl and enhancing EGFR ubiquitylation. In contrast, expression of a catalytically inactive Shp2 mutant reversed the effect of ITSN1 on Spry2 dephosphorylation and decreased Cbl-mediated EGFR ubiquitylation. In addition, disruption of ITSN1 binding to Spry2 through point mutation of the Pro-rich ITSN1 binding site in Spry2 resulted in decreased Shp2-Spry2 interaction and enhanced Spry2 tyrosine phosphorylation. This study demonstrates that ITSN1 enhances Cbl activity, in part, by modulating the interaction of Cbl with Spry2 through recruitment of Shp2 phosphatase to the Cbl-Spry2 complex. These findings reveal a new level of complexity in the regulation of RTKs by Cbl through ITSN1 binding with Shp2 and Spry2.     

Intersectin 1 enhances Cbl ubiquitylation of epidermal growth factor receptor through regulation of Sprouty2-Cbl interaction

Ubiquitylation of receptor tyrosine kinases plays a critical role in regulating the trafficking and lysosomal degradation of these important signaling molecules. We identified the multidomain scaffolding protein intersectin 1 (ITSN1) as an important regulator of this process (N. P. Martin et al., Mol. Pharmacol. 70:1463-1653, 2006) ITSN1 stimulates ubiquitylation of the epidermal growth factor receptor (EGFR) through enhancing the activity of the Cbl E3 ubiquitin ligase. However, the precise mechanism through which ITSN1 enhances Cbl activity was unclear. In this study, we found that ITSN1 enhances Cbl activity through disrupting the interaction of Cbl with the Sprouty2 (Spry2) inhibitory protein. We demonstrate that ITSN1 binds Pro-rich regions in both Cbl and Spry2 and that interaction of ITSN1 with Spry2 disrupts Spry2-Cbl interaction, resulting in enhanced ubiquitylation of the EGFR. Disruption of ITSN1 binding to Spry2 through point mutation of the Pro-rich ITSN1 binding site in Spry2 results in enhanced Cbl-Spry2 interaction and inhibition of receptor ubiquitylation. This study demonstrates that ITSN1 enhances Cbl activity by modulating the interaction of Cbl with Spry2. In addition, our results reveal a new level of complexity in the regulation of Cbl through the interaction with ITSN1 and Spry2.     

Biochemical basis for the requirement of kinase activity for Cbl-dependent ubiquitinylation and degradation of a target tyrosine kinase

Members of the Cbl family of ubiquitin ligases have emerged as crucial negative regulators of tyrosine kinase signaling. These proteins preferentially interact with and target activated tyrosine kinases for ubiquitinylation, thereby facilitating the lysosomal sorting of receptor tyrosine kinases or proteasomal degradation of nonreceptor tyrosine kinases. Recent work has indicated a crucial role of the target kinase activity in Cbl-dependent ubiquitinylation and degradation, but the biochemical basis for this requirement is not understood. Here, we have used the Src-family kinase Fyn, a well characterized Cbl target, to address this issue. Using defined Fyn mutants, we demonstrate that the kinase activity of Fyn is crucial for its Cbl-dependent ubiquitinylation and degradation, but a low level of ubiquitinylation and degradation of kinase-inactive Fyn mutants was consistently observed. Mutational induction of an open conformation enhanced the susceptibility of kinase-active Fyn to Cbl but was insufficient to promote the ubiquitinylation and degradation of kinase-inactive Fyn. Notably, the Cbl-dependent degradation of Fyn did not require the Fyn-mediated phosphorylation of Cbl. Finally, we show that the major determinant of the susceptibility of Fyn protein to Cbl-dependent ubiquitinylation and degradation is the extent to which it physically associates with Cbl; kinase activity of Fyn serves as a critical determinant to promote its association with Cbl, which we demonstrate is mediated by multiple protein-protein interactions. Our results strongly suggest that promotion of association with Cbl is the primary mechanism by which the kinase activity of the targets of Cbl contributes to their susceptibility to Cbl.