Huang Chien-Ling, Cheng Ju-Chien, Liao Chang-Hui, Stern Arnold, Hsieh Jer-Tsong, Wang Chi-Huei, Hsu Hsueh-Ling, Tseng Ching-Ping
Graduate Institutes of Medical Biotechnology and Natural Products, Chang Gung University, Tao-Yuan 333, Taiwan, Republic of China.
J Biol Chem. 2004 Oct 1;279(40):42279-89. doi: 10.1074/jbc.M402540200. Epub 2004 Jul 26.
Disabled-2 (DAB2) is an adapter protein that is up-reg-ulated during megakaryocytic differentiation of hematopoietic cells and is abundantly expressed in platelets. In this study, the role of DAB2 in integrin alpha(IIb)beta(3)-mediated matrix protein fibrinogen adhesion and cell signaling was investigated. In K562 cells differentiating to the megakaryocytic lineage, down-regulation of DAB2 by DAB2 small interfering RNA augmented integrin alpha(IIb)beta(3) activation and resulted in an increase in cell adhesion to fibrinogen. Ectopic expression of DAB2 reversed the DAB2 small interfering RNA effect or, by itself, decreased fibrinogen adhesion of K562 cells. Mutational analysis revealed that a DAB2 Ser(24) phosphorylation mutant (S24A) abrogated the inhibitory function of DAB2. The spatial and temporal association/interaction of DAB2 and platelet integrin alpha(IIb)beta(3) (CD61) in both megakaryocytic cells and platelets led us to examine the effect of Ser(24) phosphorylation on the interaction between DAB2 and integrin beta(3). Through cellular localization and co-immunoprecipitation analysis, we demonstrate for the first time that Ser(24) phosphorylation promotes membrane translocation of DAB2 and its subsequent interaction with integrin beta(3), thereby defining a mechanism for DAB2 in regulating integrin alpha(IIb)beta(3) activation and inside-out signaling. Consistent with the effect on fibrinogen adhesion, Ser(24) phosphorylation of DAB2 was also involved in the negative regulation of alpha(IIb)beta(3)-induced T cell factor transcriptional activity. In contrast, the S24A mutant acted like wild-type DAB2 and inhibited both beta-catenin- and plakoglobin-mediated T cell factor transactivation. Hence, DAB2 elicits distinct regulatory mechanisms in alpha(IIb)beta(3) and beta-catenin/plakoglobin signaling in a Ser(24) phosphorylation-dependent and -independent manner, respectively. These findings indicate Ser(24) phosphorylation as a molecular basis for DAB2 acting as a negative regulator in alpha(IIb)beta(3) inside-out signaling and contribute to our understanding of DAB2 in megakaryocytic differentiation and platelet function.
Disabled-2（DAB2）是一种衔接蛋白，在造血细胞向巨核细胞分化过程中上调表达，且在血小板中大量表达。在本研究中，我们探究了DAB2在整合素α（IIb）β3介导的基质蛋白纤维蛋白原黏附及细胞信号传导中的作用。在向巨核细胞系分化的K562细胞中，利用DAB2小干扰RNA下调DAB2可增强整合素α（IIb）β3的激活，并导致细胞对纤维蛋白原的黏附增加。DAB2的异位表达可逆转DAB2小干扰RNA的作用，或者其本身可降低K562细胞对纤维蛋白原的黏附。突变分析显示，DAB2丝氨酸（Ser）24磷酸化突变体（S24A）消除了DAB2的抑制功能。巨核细胞和血小板中DAB2与血小板整合素α（IIb）β3（CD61）在空间和时间上的关联/相互作用，促使我们研究Ser24磷酸化对DAB2与整合素β3相互作用的影响。通过细胞定位和共免疫沉淀分析，我们首次证明Ser24磷酸化促进DAB2的膜转位及其随后与整合素β3的相互作用，从而确定了DAB2在调节整合素α（IIb）β3激活和外向信号传导中的机制。与对纤维蛋白原黏附的影响一致，DAB2的Ser24磷酸化也参与了α（IIb）β3诱导的T细胞因子转录活性的负调控。相比之下，S24A突变体的作用类似于野生型DAB2，可抑制β-连环蛋白和桥粒斑蛋白介导的T细胞因子反式激活。因此，DAB2分别以Ser24磷酸化依赖和非依赖的方式，在α（IIb）β3和β-连环蛋白/桥粒斑蛋白信号传导中引发不同的调控机制。这些发现表明Ser24磷酸化是DAB2在α（IIb）β3外向信号传导中作为负调节因子发挥作用的分子基础，有助于我们理解DAB2在巨核细胞分化和血小板功能中的作用。
