Zona occludens 2 (ZO-2) has a dual localization. In confluent epithelia, ZO-2 is present at tight junctions (TJs), whereas in sparse proliferating cells it is also found at the nucleus. Previously we demonstrated that in sparse cultures, newly synthesized ZO-2 travels to the nucleus before reaching the plasma membrane. Now we find that in confluent cultures newly synthesized ZO-2 goes directly to the plasma membrane. Epidermal growth factor induces through AKT activation the phosphorylation of the kinase for SR repeats, serine arginine protein kinase 1, which in turn phosphorylates ZO-2, which contains 16 SR repeats. This phosphorylation induces ZO-2 entry into the nucleus and accumulation in speckles. ZO-2 departure from the nucleus requires intact S257, and stabilizing the β-O-linked N-acetylglucosylation (O-GlcNAc) of S257 with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate, an inhibitor of O-GlcNAcase, triggers nuclear exportation and proteosomal degradation of ZO-2. At the plasma membrane ZO-2 is not O-GlcNAc, and instead, as TJs mature, it becomes phosphorylated at S257 by protein kinase Cζ. This late phosphorylation of S257 is required for the correct cytoarchitecture to develop, as cells transfected with ZO-2 mutant S257A or S257E form aberrant cysts with multiple lumens. These results reveal novel posttranslational modifications of ZO-2 that regulate the intracellular fate of this protein.