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.