Magic™ Anti-DOK1 (Phospho Y398) polyclonal antibody

Integrins, which are heterodimeric (alpha and beta subunits) signal-transducer proteins, are essential for cell adhesion and migration. beta cytosolic tails (beta-CTs) of integrins interact with a number of cytosolic proteins including talin, Dok1, and 14-3-3 zeta. The formation of multiprotein complexes with beta-CTs is involved in the activation and regulation of integrins. The leukocyte-specific beta 2 integrins are essential for leukocyte trafficking, phagocytosis, antigen presentation, and proliferation. In this study, we examined the binding interactions between integrin beta 2-CT and T758-phosphorylated beta 2-CT with positive regulators talin and 14-3-3 zeta and negative regulator Dok1. Residues of the F3 domain of talin belonging to the C-terminal helix, beta-strand 5, and the adjacent loop were found to be involved in the binding interactions with beta 2-CT. The binding affinity between talin F3 and beta 2-CT was reduced when beta 2 T758 was phosphorylated, but this modification promoted 14-3-3 zeta binding. However, we were able to detect stable ternary complex formation of T758-phosphorylated beta 2-CT, talin F3, and 14-3-3 zeta that involved the repositioning of talin F3 on beta 2-CT. We showed that Dok1 binding to beta 2-CT was reduced in the presence of 14-3-3 zeta and when beta 2 T758 was phosphorylated. Based on these data, we propose a sequential model of beta 2 integrin activation involving these molecules. Our study provides for the first time insights toward beta 2 integrin activation that involves a multiprotein complex. Copyright (C) 2016 Elsevier Ltd. All rights reserved.

BCR-ABL1 is a fusion tyrosine kinase, which causes multiple types of leukemia. We used an integrated proteomic approach that includes label-free quantitative protein complex and phosphorylation profiling by mass spectrometry to systematically characterize the proximal signaling network of this oncogenic kinase. The proximal BCR-ABL1 signaling network shows a modular and layered organization with an inner core of three leukemia transformation-relevant adaptor protein complexes (Grb2/Gab2/Shc1 complex, CrkI complex and Dok1/Dok2 complex). We introduced an 'interaction directionality' analysis, which annotates static protein networks with information on the directionality of phosphorylation-dependent interactions. In this analysis, the observed network structure was consistent with a step-wise phosphorylation-dependent assembly of the Grb2/Gab2/Shc1 and the Dok1/Dok2 complexes on the BCR-ABL1 core. The CrkI complex demonstrated a different directionality, which supports a candidate assembly on the Nedd9 (Hef1, CasL) scaffold. As adaptor protein family members can compensate for each other in leukemic transformation, we compared members of the Dok and Crk protein families and found both overlapping and differential binding patterns. We identified an additional level of regulation for the CrkII protein via binding to 14-3-3 proteins, which was independent from its inhibitory phosphorylation. We also identified novel components of the inner core complexes, including the kinases Pragmin (Sgk223) and Lrrk1 (Lrrk2 paralog). Pragmin was found as a component of the CrkI complex and is a potential link between BCR-ABL1/CrkI and RhoA signaling. Lrrk1 is an unusual kinase with a GTPase domain. We detected Lrrk1 as a component of the Grb2/Gab2/Shc1 complex and found that it functionally interacts with the regulator of small GTPases Arap1 (Centd2) and possibly participates in the mitogen-activated protein kinase response to cellular stresses. This modular and phosphorylation-driven interaction network provides a framework for the integration of pleiotropic signaling effects of BCR-ABL1 toward leukemic transformation. Oncogene (2010) 29, 5895-5910; doi:10.1038/onc.2010.331; published online 9 August 2010