Jahn Thomas, Seipel Petra, Urschel Susanne, Peschel Christian, Duyster Justus
Department of Internal Medicine III, Laboratory of Leukemogenesis, Technical University of Munich, Munich, Germany.
Mol Cell Biol. 2002 Feb;22(4):979-91. doi: 10.1128/MCB.22.4.979-991.2002.
Grb10 is a member of the Grb7 family of adapter proteins lacking intrinsic enzymatic function and encodes functional domains including a pleckstrin homology (PH) domain and an SH2 domain. The role of different Grb10 splice variants in signal transduction of growth factors like insulin or insulin-like growth factor has been described as inhibitory or stimulatory depending on the presence of a functional PH and/or SH2 domain. Performing a yeast two-hybrid screen with the c-kit cytoplasmic tail fused to LexA as a bait and a mouse embryo cDNA library as prey, we found that the Grb10 SH2 domain interacted with the c-kit receptor tyrosine kinase. In the course of SCF-mediated activation of c-kit, Grb10 is recruited to the c-kit receptor in an SH2 domain- and phosphotyrosine-dependent but PH domain-independent manner. We found that Akt and Grb10 form a constitutive complex, suggesting a role for Grb10 in the translocation of Akt to the cell membrane. Indeed, coexpression studies revealed that Grb10 and c-kit activate Akt in a synergistic manner. This dose-dependent effect of Grb10 is wortmannin sensitive and was also seen at a lower level in cells in which c-kit was not expressed. Expression of a Grb10 mutant lacking the SH2 domain as well as a mutant lacking the PH domain did not influence Akt activity. Grb10-induced Akt activation was observed without increased phosphatidylinositol 3-kinase (PI3-kinase) activity, suggesting that Grb10 is a positive regulator of Akt downstream of PI3-kinase. Significantly, deficient activation of Akt by a constitutively activated c-kit mutant lacking the binding site for PI3-kinase (c-kitD814V/Y719F) could be fully compensated by overexpression of Grb10. In Ba/F3 cells, the incapacity of c-kitD814V/Y719F to induce interleukin-3 (IL-3)-independent growth could be rescued by overexpression of Grb10. In contrast, expression of the SH2 deletion mutant of Grb10 together with c-kitD814V/Y719F did not render Ba/F3 cells independent of IL-3. In summary, we provide evidence that Grb10 is part of the c-kit signaling pathway and that the expression level of Grb10 critically influences Akt activity. We propose a model in which Grb10 acts as a coactivator for Akt by virtue of its ability to form a complex with Akt and its SH2 domain-dependent translocation to the cell membrane.
Grb10是衔接蛋白Grb7家族的成员，缺乏内在酶活性，编码包括一个普列克底物蛋白同源（PH）结构域和一个SH2结构域的功能结构域。不同的Grb10剪接变体在胰岛素或胰岛素样生长因子等生长因子信号转导中的作用，根据功能性PH结构域和/或SH2结构域的存在情况，被描述为具有抑制或刺激作用。我们以与LexA融合的c-kit胞质尾作为诱饵，以小鼠胚胎cDNA文库作为猎物进行酵母双杂交筛选，发现Grb10的SH2结构域与c-kit受体酪氨酸激酶相互作用。在干细胞因子（SCF）介导的c-kit激活过程中，Grb10以一种依赖SH2结构域和磷酸酪氨酸但不依赖PH结构域的方式被招募到c-kit受体上。我们发现Akt和Grb10形成一个组成型复合物，提示Grb10在Akt向细胞膜转位中发挥作用。确实，共表达研究表明Grb10和c-kit以协同方式激活Akt。Grb10的这种剂量依赖性效应对渥曼青霉素敏感，在未表达c-kit的细胞中也能在较低水平观察到。缺乏SH2结构域的Grb10突变体以及缺乏PH结构域的突变体的表达均不影响Akt活性。观察到Grb10诱导的Akt激活，但磷脂酰肌醇3激酶（PI3激酶）活性未增加，这表明Grb10是PI3激酶下游Akt的正调节因子。值得注意的是，缺乏PI3激酶结合位点的组成型激活的c-kit突变体（c-kitD814V/Y719F）对Akt的激活缺陷可通过Grb10的过表达得到完全补偿。在Ba/F3细胞中，Grb10的过表达可挽救c-kitD814V/Y719F诱导白细胞介素-3（IL-3）非依赖性生长的无能。相反，Grb10的SH2缺失突变体与c-kitD814V/Y719F共同表达并不能使Ba/F3细胞不依赖IL-3。总之，我们提供证据表明Grb10是c-kit信号通路的一部分，并且Grb10的表达水平对Akt活性有至关重要的影响。我们提出一个模型，其中Grb10凭借其与Akt形成复合物的能力及其依赖SH2结构域向细胞膜的转位而作为Akt的共激活因子发挥作用。
