Anti-DOK1 (N-terminal) polyclonal antibody

Integrins are involved in cell migration and adhesion. A large number of proteins interact with the cytoplasmic tails of integrins. Dok1 is a negative regulator of integrin activation and it binds to the phosphorylated membrane proximal NxxY motif in a number of integrin beta tails. The beta tail of the beta(2) integrins contains a non-phosphorylatable NxxF motif. Hence it is unclear how Dok1 associates with the beta(2) integrins. We showed in this study using NMR and cell based analyses that residues Ser745 and Ser756 in the integrin beta(2) tail, which are adjacent to the NxxF motif, are required for Dok1 interaction. NMR analyses detected significant chemical shift changes and higher affinity interactions between Dok1 phospho-tyrosine binding (PTB) domain and integrin beta(2) tail peptide containing pSer756 compared to pSer745. The phosphorylated beta(2) peptide occupies the canonical ligand binding pocket of Dok1 based on the docked structure of the beta(2) tail-Dok1 PTB complex. Taken together, our data suggest an alternate phosphorylation switch in beta(2) integrins that regulates Dok1 binding. This could be important for cells of the immune system and their functions.

Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a transcription factor that promotes differentiation and cell survival in the stomach. PPAR gamma upregulates and interacts with caveolin-1 (Cav1), a scaffold protein of Ras/mitogen-activated protein kinases (MAPKs). The cytoplasmic-to-nuclear localization of PPAR gamma is altered in gastric cancer (GC) patients, suggesting a so-far-unknown role for Cav1 in spatial regulation of PPAR gamma signaling. We show here that loss of Cav1 accelerated proliferation of normal stomach and GC cells in vitro and in vivo. Downregulation of Cav1 increased Ras/MAPK-dependent phosphorylation of serine 84 in PPAR gamma and enhanced nuclear translocation and ligand-independent transcription of PPAR gamma target genes. In contrast, Cav1 overexpression sequestered PPAR gamma in the cytosol through interaction of the Cav1 scaffolding domain (CSD) with a conserved hydrophobic motif in helix 7 of PPA gamma's ligand-binding domain. Cav1 cooperated with the endogenous Ras/MAPK inhibitor docking protein 1 (Dok1) to promote the ligand-dependent transcriptional activity of PPAR gamma and to inhibit cell proliferation. Ligand-activated PPAR gamma also reduced tumor growth and upregulated the Ras/MAPK inhibitors Cav1 and Dok1 in a murine model of GC. These results suggest a novel mechanism of PPAR gamma regulation by which Ras/MAPK inhibitors act as scaffold proteins that sequester and sensitize PPAR gamma to ligands, limiting proliferation of gastric epithelial cells.