Protein-protein interactions at the Golgi complex and their roles in apoptosis and mitosis.

摘要

Golgin-160, ubiquitously expressed in vertebrates, localizes to the cytoplasmic side of the Golgi complex and has a large C-terminal coiled-coil domain. The non-coiled-coil N-terminal head domain contains Golgi targeting information, a cryptic nuclear localization signal (NLS), and three aspartates targeted by caspases. Caspase cleavage of this domain generates different fragments that can translocate to the nucleus by exposing the NLS. It was found that the GCP60, a Golgi resident protein, interacted weakly with the golgin-160 head domain but had a strong interaction with a caspase-generated golgin160 fragment, golgin-160(140-311). This preferential interaction increased the Golgi retention of the golgin-160 fragment in cells overexpressing GCP60. A single cysteine residue in GCP60 (Cys-463) was identified as critical for the interaction. Mutation of this cysteine blocked the interaction in vitro and disrupted the ability to retain the golgin-160 fragment at the Golgi in cells. Cys-463 was found to be redox sensitive: in its reduced form, interaction with golgin-160 was diminished or abolished, whereas oxidation of the Cys-463 by hydrogen peroxide restored the interaction. In addition, incubation with a nitric oxide donor promoted this interaction in vitro, suggesting that nuclear translocation of golgin-160(140-311) is a highly coordinated event regulated by cleavage of golgin-1 60 and oxidation or nitrosylation of GCP60.;Two serine residues in GCP60 were identified to be phosphorylated during interphase. These serines were next to an acidic cluster forming a consensus site for protein kinase CK2, which phosphorylated GCP60 in vitro. Interestingly, GCP60 C-terminal domain was dephosphorylated during mitosis. Mutation of these residues did not affect its interaction with giantin. However, replacing the serines with aspartates residues to mimic phosphorylation enhanced its interaction with Numb, a protein involved in neuronal progenitor cell differentiation. These results suggested that phosphorylation and dephosphorylation of GCP60 C-terminal region during mitosis may be important during neurogenesis.;These findings show that GCP60 protein-protein interactions can be regulated through post-translational modifications of its highly conserved C-terminal domain. Therefore, changes produced locally at Golgi membranes due to enzymatic activities or their products in response to cellular stress could promote or disrupt such interactions through post-translational modifications.