Human furin catalyzes the proteolytic maturation of many proproteins in the exocytic and endocytic secretory pathways by cleavage at the C‐terminal side of the consensus sequence‐ArgXaaLys/ArgArg decreases ‐. Both the trans‐Golgi network (TGN) concentration and intracellular routing of furin require sequences in its 56 amino acid cytoplasmic tail. Here, we show that the furin cytoplasmic tail contains multiple trafficking signals. Localization to the TGN requires a cluster of acidic amino acids that, together with a pair of serine residues, forms a casein kinase II (CK II) phosphorylation site. We show that CK II efficiently phosphorylates these serines in vitro, and using a permeabilized cell system we provide evidence that CK II is the in vivo furin kinase. Analysis by mass spectrometry shows that, in vivo, furin exists as di‐, mono‐ and non‐phosphorylated forms. Finally, employing (i) furin constructs that mimic either non‐phosphorylated or phosphorylated furin and (ii) the phosphatase inhibitor tautomycin, we show that the phosphorylation state of the furin cytoplasmic tail modulates retrieval of the endoprotease to the TGN. Thus, routing of furin is a two‐tiered process combining a set of trafficking signals comprised of the primary amino acid sequence of the tail with its phosphorylation state.

Phorbol esters such as 12-O-tetradeconylphorbol-13-acetate (TPA)activate protein kinase C, increase Connexin43 (Cx43) phosphorylation, and decrease cell-cell communication via gap junctions in many cell types. Previous work has implicated protein kinase C (PKC) in the direct phosphorylation of Cx43 at S368, which results in a change in single channel behavior that contributes to a decrease in intercellular communication. We have examined Cx43 phosphorylation in several cell lines with an antibody specific for phosphorylated S368. We show that this antibody detects Cx43 only when it is phosphorylated at S368 and, consistent with previous results, TPA treatment causes a dramatic increase in phosphorylation at S368. However, in some cell types, the increased phosphorylation at S368 did not cause a detectable shift in migration as compared with the nonphosphorylated Cx43. Immunofluorescence showed increased S368 immunolabeling in cytoplasmic and plasma membrane structures in response to TPA. Immunoblot analysis of synchronized cells showed increased phosphorylation at S368 during S and G2/M phases of the cell cycle. S-phase cells contained more total Cx43 but assembled fewer functional gap junctional channels than G0-phase cells. Since M-phase cells also communicate poorly and contain few assembled gap junctions, phosphorylation at S368 appears to be negatively correlated with gap junction assembly. Thus, both gap junctional communication and S368 phosphorylation change during S phase and G2/M, implying that phosphorylation at S368 might play a role in key cell-cycle events.