SUMOylation of the inducible (c-Fos:c-Jun)/AP-1 transcription complex occurs on target promoters to limit transcriptional activation.

The inducible proto-oncogenic (c-Fos:c-Jun)/AP-1 transcription complex binds 12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive elements (TRE) in its target genes. It is tightly controlled at multiple levels to avoid the deleterious effects of its inappropriate activation. In particular, SUMOylation represses its transactivation capacity in transient reporter assays using constitutively expressed proteins. This led to the presumption that (c-Fos:c-Jun)/AP-1 SUMOylation would be required to turn-off transcription of its target genes, as proposed for various transcription factors. Instead, thanks to the generation of an antibody specific for SUMO-modified c-Fos, we provide here direct evidence that SUMOylated c-Fos is present on a stably integrated reporter TPA-inducible promoter at the onset of transcriptional activation and colocalizes with RNA polymerase II within chromatin. Interestingly, (c-Fos:c-Jun)/AP-1 SUMOylation limits reporter gene induction, as well as the appearance of active transcription-specific histone marks on its promoter. Moreover, non-SUMOylatable mutant (c-Fos:c-Jun)/AP-1 dimers accumulate to higher levels on their target promoter, suggesting that SUMOylation might facilitate the release of (c-Fos:c-Jun)/AP-1 from promoters. Finally, activation of GADD153, an AP-1 target gene, is also associated with a rapid increase in SUMOylation at the level of its TRE and c-Fos SUMOylation dampens its induction by TPA. Taken together, our data suggest that SUMOylation could serve to buffer transcriptional activation of AP-1 target genes.

GSK3-SCF(FBXW7) targets JunB for degradation in G2 to preserve chromatid cohesion before anaphase.

JunB, an activator protein-1 (AP-1) transcription factor component, acts either as a tumor suppressor or as an oncogene depending on the cell context. In particular, JunB is strongly upregulated in anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) where it enhances cell proliferation. Although its overexpression is linked to lymphomagenesis, the mechanisms whereby JunB promotes neoplastic growth are still largely obscure. Here, we show that JunB undergoes coordinated phosphorylation-dependent ubiquitylation during the G2 phase of the cell cycle. We characterized a critical consensus phospho-degron that controls JunB turnover and identified GSK3 and SCF(FBXW7) as, respectively, the kinase and the E3 ubiquitin ligase responsible for its degradation in G2. Pharmacological or genetic inactivation of the GSK3-FBXW7-JunB axis induced accumulation of JunB in G2/M and entailed transcriptional repression of the DNA helicase DDX11, leading to premature sister chromatid separation. This abnormal phenotype due to dysregulation of the GSK3ß/JunB/DDX11 pathway is phenocopied in ALK-positive ALCL. Thus, our results reveal a novel mechanism by which mitosis progression and chromatid cohesion are regulated through GSK3/SCF(FBXW7)-mediated proteolysis of JunB, and suggest that JunB proteolysis in G2 is an essential step in maintaining genetic fidelity during mitosis.

The PKCθ pathway participates in the aberrant accumulation of Fra-1 protein in invasive ER-negative breast cancer cells.

Fra-1 is aberrantly expressed in a large number of cancer cells and tissues, and emerging evidence suggests an important role for this Fos family protein in both oncogenesis and the progression or maintenance of many tumour types. Here, we show that the concentration of Fra-1 is high in invasive oestrogen receptor (ER)-negative (ER-) breast cancer cell lines, regardless of their Ras pathway status. All of the ER- cells express high levels of activated PKCθ, and the inhibition of PKCθ activity using RNA interference or the expression of a dominant-negative mutant results in a dramatic reduction in Fra-1 abundance. Conversely, the ectopic expression of constitutively active PKCθ leads to Fra-1 phosphorylation and accumulation in poorly invasive ER+ cells. This accumulation is due to the stabilisation of the Fra-1 protein through PKCθ signalling, whereas other members of the PKC family are ineffective. Both Ste20-related proline-alanine-rich kinase (SPAK) and ERK1/2, whose activities are upregulated by PKCθ, participate in PKCθ-driven Fra-1 stabilisation. Interestingly, their relative contributions appear to be different depending on the cell line studied. ERK1/2 signalling has a major role in ER- MDA-MB-231 cells, whereas Fra-1 accumulation occurs mainly through SPAK signalling in ER- BT549 cells. Fra-1 mutational analysis shows that the phosphorylation of S265, T223 and T230 is critical for PKCθ-driven Fra-1 stabilisation. Phosphorylation of the protein was confirmed using specific antisera against Fra-1 phosphorylated on T223 or S265. In addition, Fra-1 participates in PKCθ-induced cell invasion and is necessary for PKCθ-induced cell migration. In summary, we identified PKCθ signalling as an important regulator of Fra-1 accumulation in ER- breast cancer cells. Moreover, our results suggest that PKCθ could participate in progression of some breast cancers and could be a new therapeutic target.

Fra-1 controls motility of bladder cancer cells via transcriptional upregulation of the receptor tyrosine kinase AXL.

Fos-related antigen 1 (Fra-1) is a Fos family member overexpressed in several types of human cancers. Here, we report that Fra-1 is highly expressed in the muscle-invasive form of the carcinoma of the bladder (80%) and to a lesser extent in superficial bladder cancer (42%). We demonstrate that in this type of cancer Fra-1 is regulated via a C-terminal instability signal and C-terminal phosphorylation. We show that manipulation of Fra-1 expression levels in bladder cancer cell lines affects cell morphology, motility and proliferation. The gene coding for AXL tyrosine kinase is directly upregulated by Fra-1 in bladder cancer and in other cell lines. Importantly, our data demonstrate that AXL mediates the effect of Fra-1 on tumour cell motility but not on cell proliferation. We suggest that AXL may represent an attractive therapeutic target in cancers expressing high Fra-1 levels.

Heterodimerization with Fra-1 cooperates with the ERK pathway to stabilize c-Jun in response to the RAS oncoprotein.

Multiple tumorigenic pathways converge on the activating protein-1 (AP-1) family of dimeric transcription complexes by affecting transcription, mRNA decay, posttranslational modifications, as well as stability of its JUN and FOS components. Several mechanisms have been implicated in the phosphorylation- and ubiquitylation-dependent control of c-Jun protein stability. Although its dimer composition has a major role in the regulation of AP-1, little is known about the influence of heterodimerization partners on the half-life of c-Jun. The FOS family member Fra-1 is overexpressed in various tumors and cancer cell lines wherein it controls motility, invasiveness, cell survival and cell division. Oncogene-induced accumulation of Fra-1 results from both increased transcription and phosphorylation-dependent stabilization of the protein. In this report, we describe a novel role of Fra-1 as a posttranslational regulator of c-Jun. By using both constitutively and inducible transformed rat thyroid cell lines, we found that c-Jun is stabilized in response to RAS oncoprotein expression. This stabilization requires the activity of the extracellular signal-related kinase (ERK) pathway, along with c-Jun heterodimerization with Fra-1. In particular, heterodimerization with Fra-1 inhibits c-Jun breakdown by a mechanism dependent on the phosphorylation of the Fra-1 C-terminal domain that positively controls the stability of the protein in response to ERK signaling. Therefore, Fra-1 modulates AP-1 dimer composition by promoting the accumulation of c-Jun in response to oncogenic RAS signaling.

Heat shock protein 27 is involved in SUMO-2/3 modification of heat shock factor 1 and thereby modulates the transcription factor activity.

Heat shock protein 27 (HSP27) accumulates in stressed cells and helps them to survive adverse conditions. We have already shown that HSP27 has a function in the ubiquitination process that is modulated by its oligomerization/phosphorylation status. Here, we show that HSP27 is also involved in protein sumoylation, a ubiquitination-related process. HSP27 increases the number of cell proteins modified by small ubiquitin-like modifier (SUMO)-2/3 but this effect shows some selectivity as it neither affects all proteins nor concerns SUMO-1. Moreover, no such alteration in SUMO-2/3 conjugation is achievable by another HSP, such as HSP70. Heat shock factor 1 (HSF1), a transcription factor responsible for HSP expression, is one of the targets of HSP27. In stressed cells, HSP27 enters the nucleus and, in the form of large oligomers, binds to HSF1 and induces its modification by SUMO-2/3 on lysine 298. HSP27-induced HSF1 modification by SUMO-2/3 takes place downstream of the transcription factor phosphorylation on S303 and S307 and does not affect its DNA-binding ability. In contrast, this modification blocks HSF1 transactivation capacity. These data show that HSP27 exerts a feedback inhibition of HSF1 transactivation and enlighten the strictly regulated interplay between HSPs and HSF1. As we also show that HSP27 binds to the SUMO-E2-conjugating enzyme, Ubc9, our study raises the possibility that HSP27 may act as a SUMO-E3 ligase specific for SUMO-2/3.

Identification of a C-terminal tripeptide motif involved in the control of rapid proteasomal degradation of c-Fos proto-oncoprotein during the G(0)-to-S phase transition.

c-Fos proto-oncoprotein is rapidly and transiently expressed in cells undergoing the G(0)-to-S phase transition in response to stimulation for growth by serum. Under these conditions, the rapid decay of the protein occurring after induction is accounted for by efficient recognition and degradation by the proteasome. PEST motifs are sequences rich in Pro, Glu, Asp, Ser and Thr which have been proposed to constitute protein instability determinants. c-Fos contains three such motifs, one of which comprises the C-terminal 20 amino acids and has already been proposed to be the major determinant of c-Fos instability. Using site-directed mutagenesis and an expression system reproducing c-fos gene transient expression in transfected cells, we have analysed the turnover of c-Fos mutants deleted of the various PEST sequences in synchronized mouse embryo fibroblasts. Our data showed no role for the two internal PEST motifs in c-Fos instability. However, deletion of the C-terminal PEST region led to only a twofold stabilization of the protein. Taken together, these data indicate that c-Fos instability during the G0-to-S phase transition is governed by a major non-PEST destabilizer and a C-terminal degradation-accelerating element. Further dissection of c-Fos C-terminal region showed that the degradation-accelerating effect is not contributed by the whole PEST sequence but by a short PTL tripeptide which cannot be considered as a PEST motif and which can act in the absence of any PEST environment. Interestingly, the PTL motif is conserved in other members of the fos multigene family. Nevertheless, its contribution to protein instability is restricted to c-Fos suggesting that the mechanisms whereby the various Fos proteins are broken down are, at least partially, different. MAP kinases-mediated phosphorylation of two serines close to PTL, which are both phosphorylated all over the G(0)-to-S phase transition, have been proposed by others to stabilize c-Fos protein significantly. We, however, showed that the PTL motif does not exert its effect by counteracting a stabilizing effect of these phosphorylations under our experimental conditions.

Improvement of porphyrin cellular delivery and activity by conjugation to a carrier peptide.

The chemical nuclease metalloporphyrin (manganese(III) porphyrin) can cleave DNA irreversibly and can thus constitute a potential antitumor drug. However, these molecules show low permeability to cell surface membranes. We report here the conjugation of an amphipathic carrier peptide to improve considerably its cellular delivery. The metalloporphyrin-peptide conjugate can be internalized by cells within only 5 min of incubation with a yield as high as 80%. Furthermore, the metalloporphyrin-peptide conjugate is able to cleave in vitro high or low molecular weight DNA to the same extend as metalloporphyrin alone without affecting the sequence-specific cleaving activity of the porphyrin. The conjugate is 100-fold more efficient at inducing tumor cells death than the free metalloporphyrin via a mechanism involving genomic DNA cleavage. The results are promising for further therapeutic applications with antitumor drugs such as metalloporphyrin, and also with other existing drugs by using a carrier peptide system in order to improve the cellular uptake of such molecules.

Binding of human immunodeficiency virus type 1 gp120 to CXCR4 induces mitochondrial transmembrane depolarization and cytochrome c-mediated apoptosis independently of Fas signaling.

Apoptosis of CD4(+) T lymphocytes, induced by contact between human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp120) and its receptors, could contribute to the cell depletion observed in HIV-infected individuals. CXCR4 appears to play an important role in gp120-induced cell death, but the mechanisms involved in this apoptotic process remain poorly understood. To get insight into the signal transduction pathways connecting CXCR4 to apoptosis following gp120 binding, we used different cell lines expressing wild-type CXCR4 and a truncated form of CD4 that binds gp120 but lacks the ability to transduce signals. The present study demonstrates that (i) the interaction of cell-associated gp120 with CXCR4-expressing target cells triggers a rapid dissipation of the mitochondrial transmembrane potential resulting in the cytosolic release of cytochrome c from the mitochondria to cytosol, concurrent with activation of caspase-9 and -3; (ii) this apoptotic process is independent of Fas signaling; and (iii) cooperation with a CD4 signal is not required. In addition, following coculture with cells expressing gp120, a Fas-independent apoptosis involving mitochondria and caspase activation is also observed in primary umbilical cord blood CD4(+) T lymphocytes expressing high levels of CXCR4. Thus, this gp120-mediated apoptotic pathway may contribute to CD4(+) T-cell depletion in AIDS.

Human cyclin C protein is stabilized by its associated kinase cdk8, independently of its catalytic activity.

Cyclin C belongs to the cyclin family of proteins that control cell cycle transitions through activation of specific catalytic subunits, the cyclin-dependent kinases (CDKs). However, there is as yet no evidence for any role of cyclin C and its partner, cdk8, in cell cycle regulation. Rather, the cyclin C-cdk8 complex was found associated with the RNA polymerase II transcription machinery. The periodic degradation of bona fide cyclins is crucial for cell-cycle progression and depends on the catalytic activity of the associated CDK. Here we show that endogenous cyclin C protein is quite stable with a half-life of 4 h. In contrast, exogenously expressed cyclin C is very unstable (half-life 15 min) and degraded by the ubiquitin-proteasome pathway. Co-expression with its associated cdk, however, strongly stabilizes cyclin C and results in a protein half-life near that of endogenous cyclin C. In stark contrast to data reported for other members of the cyclin family, both catalytically active and inactive cdk8 induce cyclin C stabilization. Moreover, this stabilization is accompanied in both cases by phosphorylation of the cyclin, which is not detectable when unstable. Our results indicate that cyclin C has apparently diverged from other cyclins in the regulation of its stability by its CDK partner.

Cellular and viral Fos proteins are degraded by different proteolytic systems.

c-Fos proto-oncoprotein is a short-lived transcription factor degraded by the proteasome in vivo. Its mutated forms expressed by the mouse osteosarcomatogenic retroviruses, FBJ-MSV and FBR-MSV, are stabilized two- and threefold, respectively. To elucidate the mechanisms underlying v-Fos(FBJ) and v-Fos(FBR) protein stabilization, we conducted a genetic analysis in which the half-lives and the sensitivities to various cell-permeable protease inhibitors of a variety of cellular and viral protein mutants were measured. Our data showed that the decreased degradation of v-Fos(FBJ) and v-Fos(FBR) is not simply explained by the deletion of a c-Fos destabilizing C-terminal domain. Rather, it involves a complex balance between opposing destabilizing and stabilizing mutations which are distinct and which include virally-introduced peptide motifs in both cases. The mutations in viral Fos proteins conferred both total insensitivity to proteasomal degradation and sensitivity to another proteolytic system not naturally operating on c-Fos, explaining the limited stabilization of the two proteins. This observation is consistent with the idea that FBR-MSV and FBJ-MSV expression machineries have evolved to ensure controlled protein levels. Importantly, our data illustrate that the degradation of unstable proteins does not necessarily involve the proteasome and provide support to the notion that highly related proteins can be broken down by different proteolytic systems in living cells.

Degradation of cellular and viral Fos proteins.

c-Fos proto-oncoprotein is a short-lived transcription factor with oncogenic potential. We have shown that it is massively degraded by the proteasome in vivo under various experimental conditions. Other proteolytic systems including lysosomes and calpains, might, however, also marginally operate on it. Although there is evidence that c-Fos can be ubiquitinylated in vitro, the unambiguous demonstration that ubiquitinylation is necessary for its addressing to the proteasome in vivo is still lacking. c-Jun, one of the main dimerization partners of c-Fos within the AP-1 transcription complex, is also an unstable protein. Its degradation is clearly proteasome- and ubiquitin-dependent in vivo. Interestingly, several lines of evidence indicate that the addressing of c-Fos and c-Jun to the proteasome is, at least in part, governed by different mechanisms. c-Fos has been transduced by two murine osteosarcomatogenic retroviruses under mutated forms which are more stable and more oncogenic. The stabilization is not simply accounted for by simple deletion of c-Fos main destabilizer but, rather, by a complex balance between opposing destabilizing and stabilizing mutations. Though mutations in viral Fos proteins confer full resistance to proteasomal degradation, stabilization is limited because mutations also entail sensitivity to an unidentified proteolytic system. This observation is consistent with the idea that Fos-expressing viruses have evolved to ensure control protein levels to avoid high protein accumulation-linked apoptosis. In conclusion, the unveiling of the complex mechanism network responsible for the degradation of AP-1 family members is still at its beginning and a number of issues regarding the regulation of this process and the addressing to the proteasome are still unresolved.

Sustained systemic delivery of monoclonal antibodies by genetically modified skin fibroblasts.

In vivo production and systemic delivery of therapeutic antibodies by engineered cells might advantageously replace injection of purified antibodies for treating a variety of life-threatening diseases, including cancer, acquired immunodeficiency syndrome, and autoimmune diseases. We report here that skin fibroblasts retrovirally transduced to express immunoglobulin genes can be used for sustained long-term systemic delivery of cloned antibodies in immunocompetent mice. Importantly, no anti- idiotypic response against the ectopically expressed model antibody used in this study was observed. This supports the notion that skin fibroblasts can potentially be used in antibody-based gene/cell therapy protocols without inducing any adverse immune response in treated individuals.

Efficient cell infection by Moloney murine leukemia virus-derived particles requires minimal amounts of envelope glycoprotein.

Retrovirus entry into cells is mediated by specific interactions between the retrovirally encoded Env envelope glycoprotein and a host cell surface receptor. Though a number of peptide motifs responsible for the structure as well as for the binding and fusion activities of Env have been identified, only a few quantitative data concerning the infection process are available. Using an inducible expression system, we have expressed various amounts of ecotropic and amphotropic Env at the surfaces of Moloney murine leukemia virus-derived vectors and assayed for the infectivity of viral particles. Contrary to the current view that numerous noncooperative Env-viral receptor interactions are required for cell infection, we report here that very small amounts of Env are sufficient for optimal infection. However, increasing Env density clearly accelerates the rate at which infectious attachment to cells occurs. Moreover, our data also show that a surprisingly small number of Env molecules are sufficient to drive infection, albeit at a reduced efficiency, and that, under conditions of low expression, Env molecules act cooperatively. These observations have important consequences for our understanding of natural retroviral infection as well as for the design of cell-targeted infection techniques involving retroviral vectors.

Immunotherapy of a viral disease by in vivo production of therapeutic monoclonal antibodies.

Continuous and sustained in vivo production of monoclonal antibodies by engineered cells might render long-term antibody-based treatments cost-effective, avoid side effects associated with infusion of massive doses of antibody, and circumvent possible antiidiotypic responses against the therapeutic agent. The FrCasE retrovirus induces a lethal neurodegeneration on infection of newborn mice. We report here that implantation of cellulose sulfate capsules containing cells secreting an ectopic monoclonal antibody neutralizing FrCasE can prevent animals from developing the disease. All treated mice showed reduced or undetectable viremia in addition to a lack of the histopathological lesions characteristic of FrCasE infection. This work paves the way for a novel gene/cell antibody-based immunotherapy of a variety of severe viral and nonviral diseases.

Molecular characterization of the thermosensitive E1 ubiquitin-activating enzyme cell mutant A31N-ts20. Requirements upon different levels of E1 for the ubiquitination/degradation of the various protein substrates in vivo.

According to our current knowledge, protein ubiquitination involves three steps: activation of ubiquitin through formation of an energy-rich bond with an E1 ubiquitin-activating enzyme; and transfer of activated ubiquitin onto E2 ubiquitin-conjugating enzymes, which, in turn, alone, or in combination with E3 ubiquitin-protein ligase enzymes, transfer ubiquitin onto target proteins. A31N-ts20 cells are mouse embryo fibroblasts, thermosensitive for E1. We show here that: (a) the enzymatic activity of the enzyme is heat-inactivatable in vitro; and (b) a major mechanism responsible for E1 inactivation in vivo consists of accelerated destruction. Surprisingly, a >90% reduction in E1 abundance little alters the formation of the bulk of protein-ubiquitin conjugates when A31N-ts20 cells are grown at the nonpermissive temperature, indicating that cautious interpretation of results is required when studying ubiquitination of specific substrates using this cell line. Surprisingly, our data also indicate that, in vivo, ubiquitination of the various protein substrates in A31N-ts20 cells requires different amounts of E1, indicating that this mutant cell line can be used for unveiling the existence of differences in the intimate mechanisms responsible for the ubiquitination of the various cell proteins in vivo, and for providing criteria of reliability when developing in vitro ubiquitination assays for specific proteins.

Antiviral activity of an intracellularly expressed single-chain antibody fragment directed against the murine leukemia virus capsid protein.

We have addressed the possibility that intracellularly expressed miniantibodies directed against the viral capsid protein can be used as antiretroviral agents in gene transfer experiments. R187 is a rat monoclonal antibody that has been reported to recognize the MuLV p30gag capsid polypeptide. We report here that it also binds to the Pr65gag precursor polyprotein. R187 has been cloned and expressed in the form of a single-chain variable fragment (scFv) that shows the same binding specificity as the parental antibody. When expressed intracellularly, the R187 scFv favors the production of viral particles showing reduced infectivity. It, however, exerts no detectable protective effect against infection. This was observed both when using replication-incompetent MuLV-derived vector and replication-competent wild-type MuLV. Although the intimate mechanism of the inhibition is not clear, this work raises the possibility that gene engineering of anti-capsid protein scFvs may offer an additional lead for gene therapy of severe retrovirus-linked diseases.

Caspase-dependent apoptosis of cells expressing the chemokine receptor CXCR4 is induced by cell membrane-associated human immunodeficiency virus type 1 envelope glycoprotein (gp120).

Human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins interact with CD4 and chemokine receptors on T cells to deliver signals that trigger either activation, anergy, or apoptosis. However, the molecular mechanisms driving these responses remain poorly understood. In this study we demonstrate that apoptosis is induced upon HIV-1 envelope binding to the chemokine receptor CXCR4. Cells expressing a mutant form of CXCR4 with a C-terminal deletion were also sensitive to HIV-1 envelope-mediated apoptosis, indicating that the cytoplasmic tail of CXCR4 is not required to induce the apoptotic pathway. The specificity of this process was analyzed using several inhibitors of gp120-CD4-CXCR4 interaction. Monoclonal antibodies directed against the gp120-binding site on CD4 (ST4) and against CXCR4 (MAB173) prevented the apoptotic signal in a dose-dependent manner. The cell death program was also inhibited by SDF-1alpha, the natural ligand of CXCR4, and by suramin, a G protein inhibitor that binds with a high affinity to the V3 loop of HIV-1 gp120 envelope protein. These results highlight the role played by gp120-binding on CXCR4 to trigger programmed cell death. Next, we investigated the intracellular signal involved in gp120-induced apoptosis. This cell death program was insensitive to pertussis toxin and did not involve activation of the stress- and apoptosis-related MAP kinases p38(MAPK) and SAPK/JNK but was inhibited by a broad spectrum caspase inhibitor (z-VAD.fmk) and a relatively selective inhibitor of caspase 3 (z-DEVD.fmk). Altogether, our results demonstrate that HIV induces a caspase-dependent apoptotic signaling pathway through CXCR4.

The sensitivity of c-Jun and c-Fos proteins to calpains depends on conformational determinants of the monomers and not on formation of dimers.

Milli- and micro-calpains are ubiquitous cytoplasmic cysteine proteases activated by calcium. They display a relatively strict specificity for their substrates which they usually cleave at only a limited number of sites. Motifs responsible for recognition by calpains have not been characterized yet, and recently a role for PEST motifs in this process has been ruled out. c-Fos and c-Jun transcription factors are highly sensitive to calpains in vitro. They thus provide favourable protein contexts for studying the structural requirements for recognition and degradation by these proteases. Using in vitro degradation assays and site-directed mutagenesis, we report here that susceptibility to calpains is primarily determined by conformational determinants of the monomers and not by the quaternary structure of c-Fos and c-Jun proteins. The multiple cleavage sites borne by both proteins can be divided into at least two classes of sensitivity, the most sensitive ones being easily visualized in the presence of rate-limiting amounts of calpains. One site located at position 90-91 in c-Fos protein is extremely sensitive. However, efficient proteolysis did not have any strict dependence on the nature of the amino acids on either side of the scissile bond in the region extending from P2 to P'2. The structural integrity of the monomers is not crucial for recognition by calpains. Rather, sensitive sites can be recognized independently and their recognition is dependent on the local conformation of peptide regions that may span several tens of amino acids and maybe more in the case of the identified c-Fos hypersensitive site.