Inhibitor‐of‐apoptosis proteins (IAPs), including neuronal apoptosis inhibitory protein (NAIP), inhibit cell death. Other IAPs inhibit key caspase proteases which effect cell death, but the mechanism by which NAIP acts is unknown. Here we report that NAIP, through its third baculovirus inhibitory repeat domain (BIR3), binds the neuron‐restricted calcium‐binding protein, hippocalcin, in an interaction promoted by calcium. In neuronal cell lines NSC‐34 and Neuro‐2a, over‐expression of the BIR domains of NAIP (NAIP‐BIR1–3) counteracted the calcium‐induced cell death induced by ionomycin and thapsigargin. This protective capacity was significantly enhanced when NAIP‐BIR1–3 was co‐expressed with hippocalcin. Over‐expression of the BIR3 domain or hippocalcin alone did not substantially enhance cell survival, but co‐expression greatly increased their protective effects. These data suggest synergy between NAIP and hippocalcin in facilitating neuronal survival against calcium‐induced death stimuli mediated through the BIR3 domain. Analysis of caspase activity after thapsigargin treatment revealed that caspase‐3 is activated in NSC‐34, but not Neuro‐2a, cells. Thus NAIP, in conjunction with hippocalcin, can protect neurons against calcium‐induced cell death in caspase‐3‐activated and non‐activated pathways.

The ERM protein family members ezrin, radixin, and moesin are cytoskeletal effector proteins linking actin to membrane-bound proteins at the cell surface. Here we report on the cloning of myosin regulatory light chain interacting protein (MIR), a protein with an ERMhomology domain and a carboxyl-terminal RING finger, that is expressed, among other tissues, in brain. MIR is distributed in cultured COS cells, in a punctated manner as shown using enhanced green fluorescent protein (EGFP)-tagged MIR and by staining with a specific antibody for MIR. In the yeast two-hybrid system and in transfected COS cells, MIR interacts with myosin regulatory light chain B, which in turn regulates the activity of the actomyosin complex. Overexpression of MIR cDNA in PC12 cells abrogated neurite outgrowth induced by nerve growth factor (NGF) without affecting TrkA signaling. The results show that MIR, a novel ERMlike protein, affects cytoskeleton interactions regulating cell motility, such as neurite outgrowth.Dynamic changes in cell shape and movements involve interactions between proteins in the cytoskeleton and the plasma membrane. Ezrin, radixin, moesin, and the related tumor suppressor merlin link the actin cytoskeleton to membrane-bound proteins located at membrane extension sites, such as microvilli, membrane rufflings, and at cell-cell contacts (1-3). ERM 1 proteins are also involved in cell adhesion, influencing the redistribution of various cell adhesion molecules, such as ICAM-1 (4, 5). The amino-terminal domain of ERM proteins, comprising about 300 amino acids, is conserved among the different proteins and merlin and exhibits a high degree of sequence similarity (1-3). This amino terminus is related to proteins in the large band 4.1 protein superfamily (6), including some protein tyrosine phosphatases (7) and talin (8), and it is thought to bind to membrane proteins. In contrast, the interaction of ERM proteins with F-actin seems to involve both the amino-and carboxyl-terminal region (5). Merlin, however, lacks the F-actin binding site in the carboxyl terminus, but binds it in a different manner (9).ERM proteins are present in a wide variety of tissues, notably in epithelial cells in culture (10). In contrast to other tissues, less is known about the function of ERM proteins in the nervous system. During development and regeneration, neurons grow over large distances in a process controlled by extrinsic factors and cytoskeleton interactions. Some ERM proteins have been found in the growth cones of neuronal cells (11,12), suggesting an effect on neurite outgrowth. Indeed, recently it was shown by an antisense approach, that radixin and moesin are important for growth cone morphology and motility (13). Although important for dynamic interactions between cytoskeleton and membranes, the mechanism by which ERM proteins mediate their effects on cell motility is not fully understood (5). In this study, we have identified a novel ERM-like protein, called myosin regulatory light chain interacting protein (MIR), that binds to the...

Interactions between the cell membrane and the cytoskeleton play a major role in different aspects of cell differentiation, such as cell motility, cell division, and establishment of cellular architecture. Of particular importance in this context is the association of cortical actin with the cell membrane. The ERM 1 proteins, ezrin, radixin, and moesin, are members of the large 4.1 protein family and are involved in membrane-cytoskeleton interactions. They link the actin cytoskeleton to membranebound proteins located at different membrane sites, i.e. microvilli, membrane ruffles, and cell-cell contacts (1-3). This is accomplished by binding of the C-terminal part of the ERM proteins to actin and of the N-terminal FERM domain to specific membrane proteins (4). FERM domains have been found in many different proteins and are thought to be modules for protein-protein and protein-membrane interactions (5). The ERM proteins are involved in cell adhesion and signal transduction events through phosphorylation (6, 7) and interaction with phosphoinositides (8). The ERM proteins play an important role in the activation of Rho family proteins and can interact both downstream (9) and upstream (10) of Rho.In addition, the ERM proteins are involved in membrane dynamics (11). When ezrin, radixin, and moesin were simultaneously inactivated by antisense treatment in epithelial cells, cell-cell and cell-substratum adhesion was altered (12). Double suppression of radixin and moesin, but not ezrin and radixin or moesin, alters growth cone motility, inhibiting neurite extension (13). In contrast, overexpression of ezrin in insect cells leads to enhanced cell adhesion (14). The mechanisms behind these effects are, however, not well understood, but ERM proteins are known to interact with various proteins such as CD44 and ICAM-1, -2, and -3, which helps in establishing membrane specializations (for review, see Ref. 7). The identification and characterization of further binding partners for ERM proteins can give new insights into the function of these proteins.We have recently identified a novel ERM family protein, MIR, which has an ERM domain at the N terminus and lacks actin binding, instead possessing a RING domain in the Cterminal region (15). Overexpression of MIR abrogated neurite outgrowth in PC12 cells, an effect that may be brought about by its interaction with the myosin regulatory light chain (MRLC). We describe here a novel protein interacting with MIR, called MIR-interacting saposin-like protein (MSAP), which stimulates neurite outgrowth. Sequence comparisons showed that MSAP contains a saposin domain, which is found, among others, in the sphingolipid activator proteins, the saposins (16), and in some saposin-like proteins with a similar structure (17). The effects of MSAP on neurite outgrowth were reduced by MIR. MIR was shown to induce a decrease in the levels of MRLC, which could be blocked by overexpression of MSAP or by inhibition of proteasome activity. Evidence was obtained that the decrease in MRLC levels by MIR invo...

Members of the Bcl-2 family are major regulators of cell death and survival. Bcl-2 has been shown to heterodimerize with the death-inducing protein Bax, but the mechanism of action of Bcl-2 is not fully understood. Here we show, using the human NT-2 neuronal cell line, that overexpression of Bcl-2 leads to dramatic down-regulation of the cysteine proteases ICH and CPP32/Yama, which are directly involved in cell death. In addition, the nuclear enzyme poly(ADP-ribose) polymerase was cleaved in control cells but not in cells overexpressing Bcl-2 following induction of apoptosis. The mRNA levels of ICH and CPP32/Yama were differentially affected by Bcl-2 overexpression, suggesting both transcriptional and post-transcriptional effects of the protein. These results demonstrate novel mechanisms of action of Bcl-2 in influencing the expression of death effectors such as the cysteine proteases. The relative levels of Bcl-2 and of various cysteine proteases ultimately determine survival and death of different cells, including neurons.

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