Kohji Noguchi

Actin cleavage by CPP-32/apopain during the development of apoptosis

Interleukin-1β-converting enzyme (ICE)/ced-3 family proteases play key roles in apoptosis. However, cellular substrates for ICE family proteases involved in apoptosis are not well understood. We previously showed that actin is cleaved in vitro by an ICE family protease, distinct from ICE itself, which is activated during VP-16-induced apoptosis. In this report, we demonstrate that the actin-cleaving ICE-family protease in the apoptotic cell extract is the activated CPP-32/apopain. CPP-32 effectively cleaves actin protein to 15u2009kDa and 31u2009kDa fragments. Studies with an antibody raised against Gly-Gln-Val-Ile-Thr peptide, the N-terminal sequence of the cleaved 15u2009kDa actin fragment, showed that actin is also cleaved in vivo during the development of apoptosis. Moreover, Benzyloxycarbonyl-Glu-Val-Asp-CH2OC(O)-2,6,-dichlorobenzene (Z-EVD-CH2-DCB), a selective inhibitor of CPP-32(-like) protease, efficiently inhibited the cleavage of actin and the apoptosis of VP-16-treated U937 cells. Our present results indicate that actin is the substrate of CPP-32/apopain(-like) protease both in vitro and in vivo and suggest the role of actin in the control of cell growth and apoptosis.

Physical and functional interactions between Pim-1 kinase and Cdc25A phosphatase. Implications for the Pim-1-mediated activation of the c-Myc signaling pathway.

The pim-1 oncogene encodes a serine/threonine kinase (Pim-1) involved in the transduction of cytokine-triggered mitogenic signals. Pim-1 is unique in that it closely cooperates with c-Myc not only in oncogenesis, but also in apoptosis induction. However, the molecular basis of Pim-1 function remains poorly understood, largely because the downstream effector molecule(s) for Pim-1 kinase has not been identified. Here we provide several lines of evidence that Cdc25A cell cycle phosphatase, a direct transcriptional target for c-Myc, is a substrate for Pim-1 kinase and functions as an effector for Pim-1. We found that Pim-1 physically interacts with Cdc25A both in vitro and in vivoand phosphorylates Cdc25A. We also observed that Pim-1-mediated phosphorylation of Cdc25A increases its phosphatase activity. In addition, wild-type Pim-1, but not kinase-inactive Pim-1, enhanced Cdc25A-mediated cellular transformation and apoptosis. Our results indicate that Cdc25A might be a key molecule that links Pim-1 and c-Myc and that also ties Pim-1-mediated mitogenic signals to cell cycle machinery.

Inhibition by Differentiation-inducing Agents of Wild-type p53-dependent Apoptosis in HL-60 Cells

The product of the p53 tumor‐suppressor gene has been shown to function in apoptosis and cell cycle regulation. However, there is little information regarding the regulation of apoptosis in cell differentiation. We investigated the relationship between p53‐dependent apoptosis and differentiation induction using human promyelocytic leukemia HL‐60 cells transfected with pMAMneo expression vectors containing dexamethasone‐inducible wild‐type p53 (wt‐p53) cDNA inserts. Continuous exposure of the pMAMneo/wt‐p53 transfectants to 1 μM, dexamethasone for more than 24 h caused overexpression of wt‐p53 followed by cell death with morphological changes typical of apoptosis. Using the wt‐p53‐inducible HL‐60 cells, we examined the effects of differentiation inducers on the wt‐p53‐dependent apoptosis. All‐trans retinoic acid (all‐trans RA) at 1 nM or granulocyte macrophage colonystimulating factor (GM‐CSF) at 35 pM inhibited the wt‐p53‐induced apoptosis over a 42‐h treatment. The apoptosis inhibition by GM‐CSF, but not alltrans RA, was abolished by specific inhibitors of protein kinase C. These results suggest that extracellular signals involved in the differentiation induction could modulate the wt‐p53‐dependent apoptosis through protein kinase C‐dependent and independent pathways.