Caspase-3-mediated cleavage of Akt: involvement of non-consensus sites and influence of phosphorylation

Here, we show for the first time that Akt1 is cleaved in vitro at the caspase-3 consensus site DQDD(456) downward arrow SM. Our data suggest QEEE(116) downward arrow E(117) downward arrow MD, EEMD(119) downward arrow, TPPD(453) downward arrow QD and DAKE(398) downward arrow IM as novel non-consensus caspase-3 cleavage sites. More importantly, we demonstrate that phosphorylation of Akt1 modulates its cleavage in a site-specific manner: Resistance to cleavage at site DAKE(398) (within the kinase domain) in response to phosphorylation suggests a possible mechanism by which the anti-apoptotic role of Akt1 is regulated. Our result is important in biological models which rely on Akt1 for cell survival.     

Caspase activation of mammalian sterile 20-like kinase 3 (Mst3). Nuclear translocation and induction of apoptosis

Mammalian Sterile 20-like kinase 3 (Mst3), the physiological functions of which are unknown, is a member of the germinal center kinase-III family. It contains a conserved kinase domain at its NH(2) terminus, whereas there is a regulatory domain at its COOH terminus. In this study we demonstrate that endogenous Mst3 is specifically cleaved when Jurkat cells were treated with anti-Fas antibody or staurosporine and that this cleavage is inhibited by the caspase inhibitor, Ac-DEVD-CHO. Using apoptotic Jurkat cell extracts and recombinant caspases, we mapped the caspase cleavage site, AETD(313), which is at the junction of the NH(2)-terminal kinase domain and the COOH-terminal regulatory domain. Caspase-mediated cleavage of Mst3 activates its intrinsic kinase activity, suggesting that the COOH-terminal domain of Mst3 negatively regulates the kinase domain. Furthermore, proteolytic removal of the Mst3 COOH-terminal domain by caspases promotes nuclear translocation. Ectopic expression of either wild-type or COOH-terminal truncated Mst3 in cells results in DNA fragmentation and morphological changes characteristic of apoptosis. By contrast, no such changes were exhibited for catalytically inactive Mst3, implicating the involvement of Mst3 kinase activity for mediation of these effects. Collectively, these results support the notion that caspase-mediated proteolytic activation of Mst3 contributes to apoptosis.     

Primary sequence characterization of catestatin intermediates and peptides defines proteolytic cleavage sites utilized for converting chromogranin a into active catestatin secreted from neuroendocrine chromaffin cells

Catestatin is an active 21-residue peptide derived from the chromogranin A (CgA) precursor, and catestatin is secreted from neuroendocrine chromaffin cells as an autocrine regulator of nicotine-stimulated catecholamine release. The goal of this study was to characterize the primary sequences of high molecular mass catestatin intermediates and peptides to define the proteolytic cleavage sites within CgA that are utilized in the biosynthesis of catestatin. Catestatin-containing polypeptides, demonstrated by anti-catestatin western blots, of 54-56, 50, 32, and 17 kDa contained NH(2)-terminal peptide sequences that indicated proteolytic cleavages of the CgA precursor at KK downward arrow, KR downward arrow, R downward arrow, and KR downward arrow basic residue sites, respectively. The COOH termini of these catestatin intermediates were defined by the presence of the COOH-terminal tryptic peptide of the CgA precursor, corresponding to residues 421-430, which was identified by MALDI-TOF mass spectrometry. Results also demonstrated the presence of 54-56 and 50 kDa catestatin intermediates that contain the NH(2) terminus of CgA. Secretion of catestatin intermediates from chromaffin cells was accompanied by the cosecretion of catestatin (CgA(344)(-)(364)) and variant peptide forms (CgA(343)(-)(368) and CgA(332)(-)(361)). These determined cleavage sites predicted that production of high molecular mass catestatin intermediates requires cleavage at the COOH-terminal sides of paired basic residues, which is compatible with the cleavage specificities of PC1 and PC2 prohormone convertases. However, it is notable that production of catestatin itself (CgA(344)(-)(364)) utilizes more unusual cleavage sites at the NH(2)-terminal sides of downward arrow R and downward arrow RR basic residue sites, consistent with the cleavage specificities of the chromaffin granule cysteine protease "PTP" that participates in proenkephalin processing. These findings demonstrate that production of catestatin involves cleavage of CgA at paired basic and monobasic residues, necessary steps for catestatin peptide regulation of nicotinic cholinergic-induced catecholamine release.     

Caspase-mediated cleavage of the TIAM1 guanine nucleotide exchange factor during apoptosis.

Rho family GTPases Rac and Cdc42 are pivotal regulators of apoptosis in multiple cell types. However, little is known about the mechanism by which these GTPases are regulated in response to apoptotic stimuli. Here, we demonstrate that TIAM1, a Rac-specific guanine nucleotide exchange factor, is cleaved by caspases during apoptosis. TIAM1 cleavage occurs in multiple cell lines in response to diverse apoptotic stimuli such as ceramide, Fas, and serum deprivation. Processing occurs at residue 993 of TIAM1 and removes the NH(2)-terminal of TIAM's two pleckstrin homology domains, leaving a stable fragment containing the Dbl homology and COOH-terminal pleckstrin homology domains. This leads to functional inactivation of TIAM1, as determined by failure of the cleavage product to stimulate GTP loading of Rac in vivo. Furthermore, this product is defective in signaling to two independent Rac effectors, c-Jun NH(2)-terminal kinase and serum response factor. Finally, we demonstrate that in cells treated with ceramide, cleavage of TIAM1 coincided with the inactivation of endogenous Rac. These results reveal a novel mechanism for regulating guanine nucleotide exchange factor activity and GTPase-mediated signaling pathways.     

Activation of atypical protein kinase C zeta by caspase processing and degradation by the ubiquitin-proteasome system

Atypical protein kinase C zeta (PKCzeta) is known to transduce signals that influence cell proliferation and survival. Here we show that recombinant human caspases can process PKCzeta at three sites in the hinge region between the regulatory and catalytic domains. Caspase-3, -6, -7, and -8 chiefly cleaved human PKCzeta at EETD downward arrowG, and caspase-3 and -7 also cleaved PKCzeta at DGMD downward arrowG and DSED downward arrowL, respectively. Processing of PKCzeta expressed in transfected cells occurred chiefly at EETD downward arrowG and DGMD downward arrowG and produced carboxyl-terminal polypeptides that contained the catalytic domain. Epitope-tagged PKCzeta that lacked the regulatory domain was catalytically active following expression in HeLa cells. Induction of apoptosis in HeLa cells by tumor necrosis factor alpha plus cycloheximide evoked the conversion of full-length epitope-tagged PKCzeta to two catalytic domain polypeptides and increased PKCzeta activity. A caspase inhibitor, zVAD-fmk, prevented epitope-tagged PKCzeta processing and activation following the induction of apoptosis. Induction of apoptosis in rat parotid C5 cells produced catalytic domain polypeptides of endogenous PKCzeta and increased PKCzeta activity. Caspase inhibitors prevented the increase in PKCzeta activity and production of the catalytic domain polypeptides. Treatment with lactacystin, a selective inhibitor of the proteasome, caused polyubiquitin-PKCzeta conjugates to accumulate in cells transfected with the catalytic domain or full-length PKCzeta, or with a PKCzeta mutant that was resistant to caspase processing. We conclude that caspases process PKCzeta to carboxyl-terminal fragments that are catalytically active and that are degraded by the ubiquitin-proteasome pathway.     

Structural determinants of procryptdin recognition and cleavage by matrix metalloproteinase-7

The bactericidal activity of mouse Paneth cell alphadefensins, or cryptdins, is dependent on processing of cryptdin precursors (pro-Crps) by matrix metalloproteinase-7 (MMP-7) (Wilson, C. L., Ouellette, A. J., Satchell, D. P., Ayabe, T., Lopez-Boado, Y. S., Stratman, J. L., Hultgren, S. J., Matrisian, L. M., and Parks, W. C. (1999) Science 286, 113-117). To investigate the mechanisms of pro-Crp processing by this enzyme, recombinant pro-Crp4, a His-tagged chimeric pro-Crp (pro-CC), and site-directed mutant precursors of each were digested with MMP-7, and the cleavage products were analyzed by NH(2)-terminal peptide sequencing. Proteolysis of pro-Crp4 with MMP-7 activated in vitro bactericidal activity to the level of the mature Crp4 peptide by cleaving pro-Crp4 at Ser(43) downward arrow Ile(44) and Ala(53) downward arrow Leu(54) in the proregion and near the Crp4 peptide NH(2) terminus between Ser(58) downward arrow Leu(59). Because the Crp4 NH(2) terminus occurs at Gly(61), not Leu(59), MMP-7 is necessary but insufficient to complete the processing of Crp4. Crp activating proteolysis at S58 downward arrow L59 was unaffected by I44S/I44D or L54S/L54D loss-of-function mutations in pro-Crp4, and a (L59S)-pro-CC mutant was cleaved normally at Ser(43) downward arrow Val(44) and Ser(53) downward arrow Leu(54) sites but not at the peptide NH(2) terminus. C57BL/6 mice contain an abundant (L59S)-Crp4 mutant peptide with Leu(54) at its NH(2) terminus resulting from Ala(53) downward arrow Leu(54) cleavage and loss-of-function at the Ser(58) downward arrow Ser(59) cleavage site. Thus, alpha-defensins resulting from mutations at MMP-7 cleavage sites exist in mouse populations. A pro-CC substrate containing both L54S and L59S mutations resisted cleavage at Ser(43) downward arrow Val(44) completely, showing that cleavage at one or both downstream sites must precede proteolysis at Ser(43) downward arrow Val(44). These findings show that MMP-7 activation of pro-Crps can occur without proteolysis of the proregion, and prosegment fragmentation depends, at least in part, on the release of the Crp peptide from the precursor.     

The cleavage of Akt/protein kinase B by death receptor signaling is an important event in detachment-induced apoptosis.

Epithelial cells undergo death receptor-dependent apoptosis when detached from matrix, a process termed anoikis. Activation of Akt/protein kinase B (PKB) by matrix attachment protects cells from anoikis. In this study, we establish a link between anoikis and Akt/PKB-mediated survival by demonstrating that Akt/PKB is cleaved by caspases in matrix-detached epithelial cells by a mechanism that involves death receptors. Reduced levels of Akt/PKB protein were observed in detached Madin-Darby canine kidney cells relative to cells attached to collagen. Equivalent levels of Akt/PKB, however, were detected in matrix-adherent and detached cells after inhibition of caspase activity or expression of an Akt/PKB mutant (D108+119A) that is resistant to caspase cleavage. The contribution of death domain-containing proteins to Akt/PKB cleavage was evidenced by the ability of dominant negative Fas-associated death domain to restore normal levels of Akt/PKB in matrix-detached cells. Importantly, expression of a cleavage-resistant Akt/PKB mutant protected matrix-detached cells from apoptosis. These studies suggest that members of the death receptor family promote the caspase-mediated cleavage of Akt/PKB and that this event contributes to anoikis.     

The NH2-terminal domain of Golgin-160 contains both Golgi and nuclear targeting information

Golgin-160 is a member of the golgin family of Golgi-localized membrane proteins. The COOH-terminal two-thirds of golgin-160 is predicted to form a coiled-coil, with an NH(2)-terminal "head" domain. To identify the Golgi targeting information in golgin-160, full-length and deletion constructs tagged with green fluorescent protein were generated. The head domain alone was targeted to the Golgi complex in the absence of assembly with endogenous golgin-160. Further truncations from both ends of the head domain narrowed the Golgi targeting information to 85 amino acids between residues 172 and 257. Surprisingly, certain truncations of the head domain also specifically accumulated in the nucleus. Both a nuclear localization signal (masked in the full-length protein) and information for nuclear retention contributed to the nuclear localization of these truncations. Because the golgin-160 head is cleaved by caspases during apoptosis, we examined the localization of epitope-tagged proteins corresponding to all potential caspase cleavage fragments. Our data suggest that three of six fragments could be targeted to the nucleus, provided that they are released from Golgi membranes after cleavage. The finding that both Golgi and nuclear targeting information is present in the same region of golgin-160 suggests that this protein may have more than one function.     

Alphaviruses induce apoptosis in Bcl-2-overexpressing cells: evidence for a caspase-mediated, proteolytic inactivation of Bcl-2.

Bcl-2 oncogene expression plays a role in the establishment of persistent viral infection by blocking virus-induced apoptosis. This might be achieved by preventing virus-induced activation of caspase-3, an IL-1beta-converting enzyme (ICE)-like cysteine protease that has been implicated in the death effector phase of apoptosis. Contrary to this model, we show that three cell types highly overexpressing functional Bcl-2 displayed caspase-3 activation and underwent apoptosis in response to infection with alphaviruses Semliki Forest and Sindbis as efficiently as vector control counterparts. In all three cell types, overexpressed 26 kDa Bcl-2 was cleaved into a 23 kDa protein. Antibody epitope mapping revealed that cleavage occurred at one or two target sites for caspases within the amino acid region YEWD31 (downward arrow) AGD34 (downward arrow) A, removing the N-terminal BH4 region known to be essential for the death-protective activity of Bcl-2. Preincubation of cells with the caspase inhibitor Z-VAD prevented Bcl-2 cleavage and partially restored the protective activity of Bcl-2 against virus-induced apoptosis. Moreover, a murine Bcl-2 mutant having Asp31, Asp34 and Asp36 substituted by Glu was resistant to proteolytic cleavage and abrogated apoptosis following virus infection. These findings indicate that alphaviruses can trigger a caspase-mediated inactivation of Bcl-2 in order to evade the death protection imposed by this survival factor.     

A caspase cleavage fragment of p115 induces fragmentation of the Golgi apparatus and apoptosis

In mammalian cells, the Golgi apparatus undergoes extensive fragmentation during apoptosis. p115 is a key vesicle tethering protein required for maintaining the structural organization of the Golgi apparatus. Here, we demonstrate that p115 was cleaved during apoptosis by caspases 3 and 8. Compared with control cells expressing native p115, those expressing a cleavage-resistant form of p115 delayed Golgi fragmentation during apoptosis. Expression of cDNAs encoding full-length or an NH2-terminal caspase cleavage fragment of p115 had no effect on Golgi morphology. In contrast, expression of the COOH-terminal caspase cleavage product of p115 itself caused Golgi fragmentation. Furthermore, this fragment translocated to the nucleus and its expression was sufficient to induce apoptosis. Most significantly, in vivo expression of the COOH-terminal fragment in the presence of caspase inhibitors, or upon coexpression with a cleavage-resistant mutant of p115, showed that p115 degradation plays a key role in amplifying the apoptotic response independently of Golgi fragmentation.     

Nuclear localization and apoptotic regulation of an amino-terminal domain focal adhesion kinase fragment in endothelial cells

This study investigated the subcellular compartmentalization of focal adhesion kinase (FAK) fragments and their regulation during apoptosis of human umbilical vein endothelial cells. A 50 kDa NH(2)-terminal FAK fragment and a 120 kDa FAK variant were constitutively expressed and specifically found in the nuclear fraction of cells, while a 55 kDa COOH-terminal FAK fragment was only in the cytosolic fraction. FAK cleavage fragments generated during apoptosisremained in the cytosol, while p120FAK and p50 NH(2)-terminal FAK remained in the nuclear compartment. The caspase inhibitor, ZVAD-fmk, prevented the apoptosis-induced proteolysis of p125 and p120FAK, generation of the 80 kDa cleavage product, and increased expression of p50N-FAK. Western blot with phospho-specific FAK showed that nuclear p125(FAK) was phosphorylated at a significant level at Y861, while FAK phosphorylated at Y397 and Y407 was largely in the cytosol. These results indicate that FAK NH(2)- and COOH-terminal domain fragments are segregated between nuclear and cytosolic compartments in endothelial cells and suggest novel functions for the FAK NH(2)-terminal domain.     

Caspase cleavage of the nonstructural protein NS1 mediates replication of Aleutian mink disease parvovirus

Virus-induced apoptosis of infected cells can limit both the time and the cellular machinery available for virus replication. Hence, many viruses have evolved strategies to specifically inhibit apoptosis. However, Aleutian mink disease parvovirus (ADV) is the first example of a DNA virus that not only induces apoptosis but also utilizes caspase activity to facilitate virus replication. To determine the function of caspase activity during ADV replication, virus-infected cell lysates or purified ADV proteins were incubated with various purified caspases. Caspases cleaved the major nonstructural protein of ADV (NS1) at two caspase recognition sequences, whereas ADV structural proteins could not be cleaved. Importantly, the NS1 products could be identified in ADV-infected cells but were not present in infected cells pretreated with caspase inhibitors. By mutating putative caspase cleavage sites (D to E), we mapped the two cleavage sites to amino acid residues NS1:227 (INTD downward arrow S) and NS1:285 (DQTD downward arrow S). Replication of ADV containing either of these mutations was reduced 10(3)- to 10(4)-fold compared to that of wild-type virus, and a construct containing both mutations was replication defective. Immunofluorescent studies revealed that cleavage was required for nuclear localization of NS1. The requirement for caspase activity during permissive replication suggests that limitation of caspase activation and apoptosis in vivo may be a novel approach to restricting virus replication.     

Chimeras of the Flp and Cre recombinases: tests of the mode of cleavage by Flp and Cre

The Flp and Cre recombinases are members of the integrase family of tyrosine recombinases. Each protein consists of a 13 kDa NH(2)-terminal domain and a larger COOH-terminal domain that contains the active site of the enzyme. The COOH-terminal domain also contains the major determinants for the binding specificity of the recombinase to its cognate DNA binding site. All family members cleave the DNA by the attachment of a conserved nucleophilic tyrosine residue to the 3'-phosphate group at the sites of cleavage. In order to gain further insights into the determinants of the binding specificity and modes of cleavage of Flp and Cre, we have made chimeric proteins in which we have fused the NH(2)-terminal domain of Flp to the COOH-terminal domain of Cre ("Fre") and the NH(2)-terminal domain of Cre to the COOH-terminal domain of Flp ("Clp"). These chimeras have novel binding specificities in that they bind strongly to hybrid sites containing elements from both the Flp and Cre DNA targets but poorly to the native target sites.In this study we have taken advantage of the unique binding specificities of Fre and Clp to examine the mode of cleavage by Cre, Flp, Fre and Clp. We find that the COOH-terminal domain of the recombinases determines their mode of cleavage. Thus Flp and Clp cleave in trans whereas Cre and Fre cleave in cis. These results agree with the studies of Flp and with the cocrystal structure of Cre bound to its DNA target site. They disagree with our previous findings that Cre could carry out trans cleavage. We discuss the variations in the experimental approaches in order to reconcile the different results.     

Caspase Cleavage of Keratin 18 and Reorganization of Intermediate Filaments during Epithelial Cell Apoptosis

Keratins 8 (K8) and 18 (K18) are major components of intermediate filaments (IFs) of simple epithelial cells and tumors derived from such cells. Structural cell changes during apoptosis are mediated by proteases of the caspase family. During apoptosis, K18 IFs reorganize into granular structures enriched for K18 phosphorylated on serine 53. K18, but not K8, generates a proteolytic fragment during drug- and UV light–induced apoptosis; this fragment comigrates with K18 cleaved in vitro by caspase-6, -3, and -7. K18 is cleaved by caspase-6 into NH₂-terminal, 26-kD and COOH-terminal, 22-kD fragments; caspase-3 and -7 additionally cleave the 22-kD fragment into a 19-kD fragment. The cleavage site common for the three caspases was the sequence VEVD/A, located in the conserved L1-2 linker region of K18. The additional site for caspases-3 and -7 that is not cleaved efficiently by caspase-6 is located in the COOH-terminal tail domain of K18. Expression of K18 with alanine instead of serine at position 53 demonstrated that cleavage during apoptosis does not require phosphorylation of serine 53. However, K18 with a glutamate instead of aspartate at position 238 was resistant to proteolysis during apoptosis. Furthermore, this cleavage site mutant appears to cause keratin filament reorganization in stably transfected clones. The identification of the L1-2 caspase cleavage site, and the conservation of the same or very similar sites in multiple other intermediate filament proteins, suggests that the processing of IFs during apoptosis may be initiated by a similar caspase cleavage.     

Caspase-3 mediated cleavage of HsRad51 at an unconventional site.

The human recombinase HsRad51 is cleaved during apoptosis. We have earlier observed cleavage of the 41-kDa full-length protein into a 33-kDa product in apoptotic Jurkat cells and in in vitro translated HsRad51 after treatment with activated S-100 extract. In this study, site-directed mutagenesis was used for mapping of the cleavage site to AQVD274 downward arrow G, which does not correspond to a conventional caspase cleavage site. The absence of HsRad51 cleavage in staurosporine-treated apoptotic MCF-7 cells, which lack caspase-3, indicates that caspase-3 is essential for HsRad51 cleavage in vivo. Cleavage into the 33-kDa fragment was generated by recombinant caspase-3 and -7 in in vitro translated wild type HsRad51, but not in the HsRad51 AQVE274 downward arrow G mutant. Similarly, HsRad51 of Jurkat cell extracts was cleaved into the 33-kDa product by recombinant caspase-3, whereas caspase-7 failed to cleave endogenous HsRad51. The cleavage of in vitro translated wild type and AQVE274 downward arrow G mutant HsRad51 as well as of endogenous HsRad51 also gave rise to a smaller fragment, which corresponds in size to a recently reported DVLD187 downward arrow N HsRad51 cleavage product. In Jurkat cell extracts, the AQVD274 downward arrow G and DVLD187 downward arrow N cleavage products of HsRad51 appeared at equal concentrations of caspase-3. Moreover both fragments were generated by induction of apoptosis in MDA-MB 157 cells with staurosporine and in Jurkat cells with camptothecin. Thus, two sites in the HsRad51 sequence are targets for caspase cleavage both in vitro and in vivo.     

Phosphatidylinositol glycan (PI-G) anchored membrane proteins. Amino acid requirements adjacent to the site of cleavage and PI-G attachment in the COOH-terminal signal peptide.

Secreted proteins are processed from a nascent form that contains an NH2-terminal signal peptide. During processing, the latter is cleaved by a specific NH2-terminal signal peptidase. The nascent form of phosphatidylinositol glycan (PI-G) tailed proteins contain both an NH2- and a COOH-terminal signal peptide. The two signal peptides have much in common, such as size and hydrophobicity. The COOH-terminal peptide is also cleaved during processing. We propose that the amino acid in a nascent protein that ultimately combines with the PI-G moiety be designated the omega site. Amino acids adjacent and COOH-terminal to the omega site would then be omega + 1, omega + 2, etc. In previous studies, we showed that allowable substitutions at the omega site of an engineered form of placental alkaline phosphatase (miniPLAP) are limited to 6 small amino acids. In the present study, mutations were made at the omega + 1 and omega + 2 sites. At the omega + 1 site, processing to varying degrees was observed with 8 of the 9 amino acids substituted for alanine, the normal constituent. Only the proline mutant showed no processing. By contrast, the only substituents permitted at the omega + 2 site were glycine and alanine, with only trace activity observed with serine and cysteine. Thus, just as there is a -1, -3 rule for predicting cleavage by NH2-terminal signal peptidase, there appears to be a comparable omega, omega + 2 rule for predicting cleavage/PI-G addition by COOH-terminal signal transamidase.     

Caspase-3-mediated processing of poly(ADP-ribose) glycohydrolase during apoptosis

Poly(ADP-ribose) glycohydrolase (PARG) is responsible for the catabolism of poly(ADP-ribose) synthesized by poly(ADP-ribose) polymerase (PARP-1) and other PARP-1-like enzymes. In this work, we report that PARG is cleaved during etoposide-, staurosporine-, and Fas-induced apoptosis in human cells. This cleavage is concomitant with PARP-1 processing and generates two C-terminal fragments of 85 and 74 kDa. In vitro cleavage assays using apoptotic cell extracts showed that a protease of the caspase family is responsible for PARG processing. A complete inhibition of this cleavage was achieved at nanomolar concentrations of the caspase inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde, suggesting the involvement of caspase-3-like proteases. Consistently, recombinant caspase-3 efficiently cleaved PARG in vitro, suggesting the involvement of this protease in PARG processing in vivo. Furthermore, caspase-3-deficient MCF-7 cells did not show any PARG cleavage in response to staurosporine treatment. The cleavage sites identified by site-directed mutagenesis are DEID(256) downward arrow V and the unconventional site MDVD(307) downward arrow N. Kinetic studies have shown similar maximal velocity (V(max)) and affinity (K(m)) for both full-length PARG and its apoptotic fragments, suggesting that caspase-3 may affect PARG function without altering its enzymatic activity. The early cleavage of both PARP-1 and PARG by caspases during apoptosis suggests an important function for poly(ADP-ribose) metabolism regulation during this cell death process.     

Nod1, an Apaf-1-like activator of caspase-9 and nuclear factor-kappaB.

Ced-4 and Apaf-1 belong to a major class of apoptosis regulators that contain caspase-recruitment (CARD) and nucleotide-binding oligomerization domains. Nod1, a protein with an NH2-terminal CARD-linked to a nucleotide-binding domain and a COOH-terminal segment with multiple leucine-rich repeats, was identified. Nod-1 was found to bind to multiple caspases with long prodomains, but specifically activated caspase-9 and promoted caspase-9-induced apoptosis. As reported for Apaf-1, Nod1 required both the CARD and P-loop for function. Unlike Apaf-1, Nod1 induced activation of nuclear factor-kappa-B (NF-kappaB) and bound RICK, a CARD-containing kinase that also induces NF-kappaB activation. Nod1 mutants inhibited NF-kappaB activity induced by RICK, but not that resulting from tumor necrosis factor-alpha stimulation. Thus, Nod1 is a leucine-rich repeat-containing Apaf-1-like molecule that can regulate both apoptosis and NF-kappaB activation pathways.     

Processing of pulmonary surfactant protein B by napsin and cathepsin H

Surfactant protein B (SP-B) is an essential constituent of pulmonary surfactant. SP-B is synthesized in alveolar type II cells as a preproprotein and processed to the mature peptide by the cleavage of NH2- and COOH-terminal peptides. An aspartyl protease has been suggested to cleave the NH2-terminal propeptide resulting in a 25-kDa intermediate. Napsin, an aspartyl protease expressed in alveolar type II cells, was detected in fetal lung homogenates as early as day 16 of gestation, 1 day before the onset of SP-B expression and processing. Napsin was localized to multivesicular bodies, the site of SP-B proprotein processing in type II cells. Incubation of SP-B proprotein from type II cells with a crude membrane extract from napsin-transfected cells resulted in enhanced levels of a 25-kDa intermediate. Purified napsin cleaved a recombinant SP-B/EGFP fusion protein within the NH2-terminal propeptide between Leu178 and Pro179, 22 amino acids upstream of the NH2 terminus of mature SP-B. Cathepsin H, a cysteine protease also implicated in pro-SP-B processing, cleaved SP-B/EGFP fusion protein 13 amino acids upstream of the NH2 terminus of mature SP-B. Napsin did not cleave the COOH-terminal peptide, whereas cathepsin H cleaved the boundary between mature SP-B and the COOH-terminal peptide and at several other sites within the COOH-terminal peptide. Knockdown of napsin by small interfering RNA resulted in decreased levels of mature SP-B and mature SP-C in type II cells. These results suggest that napsin, cathepsin H, and at least one other enzyme are involved in maturation of the biologically active SP-B peptide.     

Proteolytic activation of protein kinase C-epsilon by caspase-mediated processing and transduction of antiapoptotic signals

Several novel protein kinase C (PKC) isozymes have been identified as substrates for caspase-3. We have previously shown that novel PKCepsilon is cleaved during apoptosis in MCF-7 cells that lack any functional caspase-3. In the present study, we show that in vitro-translated PKCepsilon is processed by human recombinant caspase-3, -7, and -9. Tumor necrosis factor-alpha (TNF) triggered processing of PKCepsilon to a 43-kDa carboxyl-terminal fragment, and cell-permeable caspase inhibitors prevented TNF-induced processing of PKCepsilon in MCF-7 cells. PKCepsilon was cleaved primarily at the SSPD downward arrow G site to generate two fragments with an approximate molecular mass of 43 kDa. It was also cleaved at the DDVD downward arrow C site to generate two fragments with molecular masses of 52 and 35 kDa. Treatment of MCF-7 cells with TNF resulted in the activation of PKCepsilon, and mutation at the SSPD downward arrow G (D383A) site inhibited proteolytic activation of PKCepsilon. Overexpression of wild-type but not dominant-negative PKCepsilon in MCF-7 cells delayed TNF-induced apoptosis, and mutation at the D383A site prevented antiapoptotic activity of PKCepsilon. These results suggest that cleavage of PKCepsilon by caspase-7 at the SSPD downward arrow G site results in the activation of PKCepsilon. Furthermore, activation of PKCepsilon was associated with its antiapoptotic function.