Wang Y, Yeung Y G, Stanley E R
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
J Cell Biochem. 1999 Jan 1;72(1):119-34.
Addition of colony stimulating factor-1 (CSF-1) to macrophages stimulates the rapid, transient tyrosine phosphorylation, membrane association and multiubiquitination of Cbl (Wang et al. [1996] J. Biol. Chem. 271:17-20). Kinetic analysis reveals that the tyrosine phosphorylation of Cbl is coincident with its plasma membrane translocation and association with the activated tyrosine phosphorylated CSF-1 R, p85, Grb2, and tyrosine phosphorylated p58Shc and that these events precede the simultaneous multiubiquitination of Cbl and the CSF-1 R. Tyrosine phosphorylation and multiubiquitination of the cell surface CSF-1 R are stoichiometric and the multiubiquitinated CSF-1 R is degraded. Similarly, the membrane associated Cbl is almost stoichiometrically ubiquitinated, but the ubiquitinated Cbl is not degraded, being recovered, deubiquitinated, in the cytosol 3-10 min after stimulation at 37 degrees C. In the membrane fraction of cells stimulated at 4 degrees C, the association of p58Shc and Grb2 with Cbl is stable, whereas its association with Sos and p85 is transient and their dissociation occurs at the time CSF-1 R and Cbl multiubiquitination commence. The membrane translocation and the pattern of association of Sos with the CSF-1R, p85, Grb2, and p58Shc resemble those of Cbl but Sos is not tyrosine phosphorylated, nor multiubiquitinated and the coprecipitation of these proteins, other than Grb2, with Sos is much less. Complexes formed by Sos and Cbl are largely independent and membrane complexes of Cbl with other tyrosine phosphorylated proteins, p85 and Grb2 also contain CSF-1 R. These data raise the possibility that the predicted negative regulatory role of Cbl in macrophages is its enhancement of ligand-induced CSF-1 R internalization/degradation.
向巨噬细胞中添加集落刺激因子-1（CSF-1）可刺激Cbl快速、短暂的酪氨酸磷酸化、膜结合及多聚泛素化（Wang等人，[1996]《生物化学杂志》271:17 - 20）。动力学分析表明，Cbl的酪氨酸磷酸化与其质膜转位以及与活化的酪氨酸磷酸化的CSF-1 R、p85、Grb2和酪氨酸磷酸化的p58Shc的结合同时发生，并且这些事件先于Cbl和CSF-1 R同时发生的多聚泛素化。细胞表面CSF-1 R的酪氨酸磷酸化和多聚泛素化是化学计量的，并且多聚泛素化的CSF-1 R会被降解。同样，膜相关的Cbl几乎以化学计量方式被泛素化，但泛素化的Cbl不会被降解，在37℃刺激后3 - 10分钟在细胞质中被回收、去泛素化。在4℃刺激的细胞的膜组分中，p58Shc和Grb2与Cbl的结合是稳定的，而其与Sos和p85的结合是短暂的，并且它们的解离发生在CSF-1 R和Cbl多聚泛素化开始之时。Sos与CSF-1R、p85、Grb2和p58Shc的膜转位及结合模式类似于Cbl，但Sos不会发生酪氨酸磷酸化，也不会多聚泛素化，并且除Grb2外，这些蛋白质与Sos的共沉淀要少得多。由Sos和Cbl形成的复合物在很大程度上是独立的，Cbl与其他酪氨酸磷酸化蛋白p85和Grb2的膜复合物中也含有CSF-1 R。这些数据增加了一种可能性，即Cbl在巨噬细胞中预测的负调控作用是其增强配体诱导的CSF-1 R内化/降解。
