S. node activated by VEGF, but not Ang-1, that specifically modulates EC proliferation during angiogenesis. The concerted action of VEGF and angiopoietin-1 (Ang-1)1 on endothelial cells (ECs) regulates the process of new blood vessel formation, Seocalcitol called angiogenesis (1). During vascular development, VEGF and Ang-1 have complementary roles to form mature blood vessels. VEGF plays a key role in vessel sprouting and initiation of new vessels, whereas Ang-1 is required for subsequent vessel maturation (2C4). Pathological angiogenesis leads to aberrant blood vessel formation in diseases such as cancer progression and metastasis or in vascular retinopathies (5, 6). Targeting intracellular signaling events elicited by VEGF and Ang-1 in ECs therefore holds promise for the treatment of angiogenic diseases (7). Through activation of their cognate tyrosine kinase receptors, VEGFR2 and Seocalcitol Tie2, VEGF and Ang-1 trigger phosphorylation of multiple intracellular effectors to induce proliferation, survival and migration of ECs (8, 9). When examined individually, it is appreciated that both receptors activate common signaling pathways in ECs such as ERK/MAPK (10, 11), PI3K/Akt (12C14), and p38 MAPK (11, 15) to induce angiogenesis. However, VEGF and Ang-1 must signal differently to cellCcell junctions to respectively augment or decrease endothelial permeability to macromolecules (16C19). This shows that, Seocalcitol Opn5 in order to induce angiogenesis, VEGF and Ang-1 must activate overlapping and diverging signaling pathways in ECs. There are numerous studies on the implication of individual intracellular signaling pathways that are activated by VEGF and Ang-1 to control angiogenesis. However, a global comparison and analysis of signaling pathways activated in ECs by these growth factors is needed to uncover novel interrelations between specific intracellular signaling events that control the angiogenic response. The endothelial junctions have long been associated with barrier functions, however they also receive and transmit signals that regulate cell communication, differentiation and proliferation (20C22). Proteins that form endothelial intercellular junctions integrate signaling events that are important for angiogenesis. For instance, genetic deletion of VE-cadherin, -catenin, or ZO-1 in mice leads to embryonic lethally because of vascular defects (23C26). In addition, it is well established that signals transmitted from intercellular junctions to the nucleus control contact-mediated inhibition of cell proliferation. In ECs, the adherens junction proteins -catenin and p120-catenin are known to elicit signaling pathways that induce proliferation when junctions are disrupted (21, 27). Both proteins can translocate to the nucleus and act as modulator of gene expression through interaction with the TCF/LEF transcription factors for -catenin or by relieving the repressor activity of the transcription factor Kaiso for p120-catenin (28, 29). The tight junction protein ZO-1 was recently shown Seocalcitol in ECs to function as a major cytoskeletal organizer that orchestrates adherens junctions to control barrier function, cell migration, and angiogenesis (30). However, the role of ZO-1 in the regulation of EC proliferation is undefined. Herein, the phosphoproteomes of ECs treated with VEGF or Ang-1 were systematically compared to profile the activation of intracellular signaling pathways. Network analysis of the phosphoproteins regulated by VEGF and Ang-1 uncovered a cluster of cell-cell junction proteins unique to VEGF treatment, which is linked to activation of MAPK1 and promotion of EC proliferation. We demonstrate that ZO-1 is the central regulator of this cluster of cell junction proteins to promote MAPK1 activation. Furthermore, we observed that reduction of the cellular levels of ZO-1 correlates with cell proliferation during retinal vascular development in mice. Collectively, our comparative phosphoproteomic analyses identified a regulatory signaling node, differentially engaged by VEGF over Ang-1, that controls EC proliferation. EXPERIMENTAL PROCEDURES Cell Culture and Reagents Bovine aortic endothelial cells (BAECs), obtained from VEC Technologies (Rensselaer, NY), were cultured in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (HyClone), 2 mm l-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin. BAECs were treated with the recombinant human VEGF-A and recombinant human Ang-1 obtained from R&D System. The primary antibodies used were: Anti-phospho-p44/42 MAPK (Thr202/Tyr204) (monoclonal antibody [mAb]), p42/44 MAPK (polyclonal antibody [pAb]), phospho-Ser1179-eNOS (pAb), eNOS (mAb), beta-actin (mAb), phospho-Ser252 p120-catenin (pAb), and BrdU.