The role of p115 vesicle tethering protein in Golgi mediated apoptosis signaling

Abstract

In mammalian cells, the Golgi apparatus consists of a series of flattened membrane cisternae located at the perinuclear region of the cell. During apoptosis, the Golgi undergoes irreversible fragmentation due to caspase mediated cleavage of its structural proteins, including the vesicle tethering protein p115. Cleavage of p115 by caspase-8 and -3 at Asp757 generates a C-terminal caspase cleavage fragment (CTF) of 205 amino acids (residues 758--961). The CTF translocates into the nucleus and its nuclear localization as well as a minimal region from residues 859--884 are required for subsequent apoptosis induction.;The mechanism by which CTF enters the nucleus and induces apoptosis is not known. Here, we show that CTF nuclear translocation is regulated by SUMOylation and that CTF-induced apoptosis is dependent on the activity of the tumor suppressor p53. Expression of the CTF induced the expression of PUMA (p53 Upregulated Modulator of Apoptosis) potentiated the levels of p53 and activated phospho-p53. The MAPK families of kinases have been shown to phosphorylate p53 in response to various stressful stimuli. Examination of the effect of MAPK inhibitors on p53 levels showed that CTF-induced p53 phosphorylation and stabilization was attenuated by the MEK1 inhibitor U0126. To understand the mechanism of CTF-mediated p53 activation, co-immunoprecipitation studies between the CTF, p53, and ERK were conducted. Both p53 and ERK co-immunoprecipitated with the CTF and that the terminal acidic domain of the CTF (Residues 931--962) is required for these interactions.;Full length p115 exists as a homodimer in its native state. The CTF is also able to form dimers and residues 859--884 previously shown to be required for apoptosis are required for dimer formation. Expression of the CTF promoted p53-ERK interaction, an effect that was abrogated with the deletion of residues 859--884. Together, our data suggest a model where CTF acts as a tether or scaffold that increases the proximity of ERK and p53 to facilitate their interaction and the subsequent phosphorylation and activation of p53. The conserved tethering function of the CTF parallels the role of full length p115 in membrane-tethering in the new context of Golgi-mediated stress and apoptosis signaling.