Bim, a "BH3-only" protein, is expressed de novo following withdrawal of serum survival factors and promotes cell death. We have shown previously that activation of the ERK1/2 pathway promotes phosphorylation of Bim EL , targeting it for degradation via the proteasome. However, the nature of the kinase responsible for Bim EL phosphorylation remained unclear. We now show that Bim EL is phosphorylated on at least three sites in response to activation of the ERK1/2 pathway. By using the peptidylprolyl isomerase, Pin1, as a probe for proline-directed phosphorylation, we show that ERK1/2-dependent phosphorylation of Bim EL occurs at (S/T)P motifs. ERK1/2 phosphorylates Bim EL , but not Bim S or Bim L , in vitro, and mutation of Ser 65 to alanine blocks the phosphorylation of Bim EL by ERK1/2 in vitro and in vivo and prevents the degradation of the protein following activation of the ERK1/2 pathway. We also find that ERK1/2, but not JNK, can physically associate with GSTBim EL , but not GST-Bim L or GST-Bim S , in vitro. ERK1/2 also binds to full-length Bim EL in vivo, and we have localized a potential ERK1/2 "docking domain" lying within a 27-amino acid stretch of the Bim EL protein. Our findings provide new insights into the post-translational regulation of Bim EL and the role of the ERK1/2 pathway in cell survival signaling.

A reduction in plasma membrane cholesterol is one of the early events that either triggers or is closely associated with capacitation of mammalian spermatozoa. In this investigation, we have examined the effects of cholesterol efflux on tyrosine phosphorylation, lipid diffusion, and raft organization in boar spermatozoa. Results show that a low level of cholesterol efflux, mediated by 5 mM methyl-beta-cyclodextrin (MBCD), enhances capacitation and induces phosphorylation of two proteins at 26 and 15 kDa without affecting sperm viability. Lipid diffusion rates under these conditions are largely unaffected except when cholesterol efflux is excessive. Low-density Triton X100-insoluble complexes (lipid rafts) were isolated from spermatozoa and found to have a restricted profile of proteins. Capacitation-associated cholesterol efflux has no effect on raft composition, but cholesterol depletion destabilizes them completely and phosphorylation is suppressed. During MBCD-mediated capacitation, the distribution of GM1 gangliosides on spermatozoa changes in a sequential manner from overlying the sperm tail to clustering on the sperm head. It is concluded that there is a safe window for removal of plasma membrane cholesterol from spermatozoa within which protein phosphorylation and polarized migration of lipid rafts take place. A preferential loss of cholesterol from the nonraft pool may be the stimulus that promotes raft clustering over the anterior sperm head.

The BH3-only protein, Bim, exists as three splice variants (Bim S , Bim L , and Bim EL ) of differing pro-apoptotic potency. Bim EL , the least effective killer, is degraded by the proteasome in response to phosphorylation by extracellular signal-regulated kinases 1 and 2 (ERK1/2). ERK1/2-dependent phosphorylation correlates with the presence of a domain unique to the Bim EL splice variant that includes the major ERK1/2 phosphorylation site Ser 65 . However, efficient phosphorylation by ERK1/2, c-Jun N-terminal kinase, or p38 requires the presence in the substrate of a discrete kinase-docking domain as well as the phosphoacceptor site. Here we show that the region unique to Bim EL (amino acids 41-97) harbors two potential DEF-type ERK1/2 kinase-docking domains, DEF1 and DEF2. Peptide competition assays revealed that the DEF2 peptide could act autonomously to bind active ERK1/2, whereas the DEF1 peptide did not. Truncation analysis identified a minimal region, residues 80 -97, containing the DEF2 motif as sufficient for ERK1/2 binding. Mutation of key residues in the DEF2 motif abolished the interaction of ERK1/2 and Bim EL and also abolished ERK1/2-dependent phosphorylation of Bim EL in vivo, thereby stabilizing the protein and enhancing cytotoxicity. Our results identify a new physiologically relevant functional motif in Bim EL that may account for the distinct biological properties of this splice variant.The BH3-only protein Bim is a pro-apoptotic member of the Bcl-2 protein family that links stress-induced signals to the core apoptotic machinery (1, 2). Expression of the Bim gene is induced at the transcriptional level in response to withdrawal of cytokines and survival factors due to inactivation of protein kinase B (3) or the ERK1/2 pathway (4). In addition, the JNK 1 (c-Jun N-terminal kinase) pathway promotes c-Jun-dependent Bim expression in neurons following the withdrawal of nerve growth factor (5, 6). Alternative splicing of the Bim gene gives rise to the short, long, and extra-long Bim proteins (Bim S , Bim L , and Bim EL ) (7), thereby introducing additional levels of regulation that may account for their differences in pro-apoptotic potency. For example, Bim S is the most effective killer and is the simplest form, consisting largely of the pro-death BH3 domain and a C-terminal membrane-tethering domain (7). Bim L contains an additional domain through which it can interact with dynein light chain 1 (DLC1) with the result that in viable cells Bim L is sequestered at microtubules and so is a less effective killer (8). Disruption of microtubules can cause the redistribution of Bim L to the mitochondria, and this may be due to JNK-dependent phosphorylation of Bim L at sites adjacent to the DLC1-binding site (9). Bim EL also contains the DLC1-binding site but is the least effective killer, and this may be explained by the fact that Bim EL protein stability is subject to post-translational regulation. Activation of the ERK1/2 pathway promotes the proteasomal degradation of Bim EL (10), and this correlates ...

The molecules on mammalian spermatozoa that mediate recognition and binding to the zona pellucida of the egg are still not understood. Current concepts favour their assembly into multimolecular complexes in the plasma membrane in response to cholesterol efflux, an important step during sperm capacitation. Here, we track in real time diffusion of cross-linked clusters containing zona-binding molecules and GM1 gangliosides in the plasma membrane of live boar spermatozoa before and after cholesterol reduction. Both GM1 gangliosides and zona-binding molecules partition into a low density Triton X100 resistant phase suggesting their association with lipid rafts. Initially, GM1 and zona-binding molecules localize to the apical ridge on the acrosome but following cholesterol efflux with methyl-beta-cyclodextrin, clusters containing zona-binding molecules diffuse randomly over the acrosomal domain. Diffusing clusters of either type do not access the postacrosome. Spermatozoa agglutinated head-to-head show contact-induced coalescence of GM1 gangliosides (but not zona-binding molecules) suggestive of a specific mechanosensitive response. Thus, cholesterol efflux initiates diffusion (and possibly formation) of novel lipid raft-like structures containing zona-binding molecules over the sperm acrosome. We hypothesise that in combination with contact coalescence, these mechanisms concentrate important molecules to the appropriate site on the sperm surface to mediate zona binding.

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