Caspase-mediated cleavage of JNK during stress-induced apoptosis

The c-Jun N-terminal kinases (JNKs) are a subfamily of the mitogen-activated protein kinases (MAPKs). The JNKs are encoded by three separate genes (jnk1, jnk2, and jnk3), which are spliced alternatively to create 10 JNK isoforms that are either p46 or p54 in size. In this study, we found that the p52 form of JNK emerged in human leukemia MOLT-4 or U937 cells following X-irradiation or heat treatment. The accumulation of p52 coincided with the reduction of p54 JNK. On the other hand, the amounts of p46 JNK did not change by X-irradiation. Induction of the p52 form of JNK also paralleled the appearance of the active form of caspase-3 and was suppressed by a caspase-specific inhibitor, Ac-DEVD-CHO, but not by Ac-YVAD-CHO. In vitro cleavage assays indicated that recombinant human JNK1beta2 and JNK2beta2 were cleaved by caspase-3, and that the mutation of aspartic acid at position 413 of JNK1beta2 or 410 of JNK2beta2 to alanine abolished the cleavage. Altogether, our results demonstrated that p54 JNKs, at least JNK1beta2 and JNK2beta2, were new selective targets of caspases in JNK splicing variants, and suggested that the p52 form could serve as a marker of apoptosis.     

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.     

Caspase-mediated cleavage of the U snRNP-associated Sm-F protein during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. The spliceosomal Sm proteins are recognized by the so-called anti-Sm autoantibodies, an antibody population found exclusively in patients suffering from systemic lupus erythematosus. We have studied the effects of apoptosis on the Sm proteins and demonstrate that one of the Sm proteins, the Sm-F protein, is proteolytically cleaved in apoptotic cells. Cleavage of the Sm-F protein generates a 9-kDa apoptotic fragment, which remains associated with the U snRNP complexes in apoptotic cells. Sm-F cleavage is dependent on caspase activation and the cleavage site has been located near the C-terminus, EEED(81) downward arrow G. Use of different caspase inhibitors suggests that besides caspase-8 other caspases are implicated in Sm-F cleavage. A C-terminally truncated mutant of the Sm-F protein, representing the modified form of the protein, is capable of forming an Sm E-F-G complex in vitro that is recognized by many anti-Sm patient sera.     

Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD³⁶⁹↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.     

Caspase-mediated cleavage of the stacking protein GRASP65 is required for Golgi fragmentation during apoptosis

The mammalian Golgi complex is comprised of a ribbon of stacked cisternal membranes often located in the pericentriolar region of the cell. Here, we report that during apoptosis the Golgi ribbon is fragmented into dispersed clusters of tubulo-vesicular membranes. We have found that fragmentation is caspase dependent and identified GRASP65 (Golgi reassembly and stacking protein of 65 kD) as a novel caspase substrate. GRASP65 is cleaved specifically by caspase-3 at conserved sites in its membrane distal COOH terminus at an early stage of the execution phase. Expression of a caspase-resistant form of GRASP65 partially preserved cisternal stacking and inhibited breakdown of the Golgi ribbon in apoptotic cells. Our results suggest that GRASP65 is an important structural component required for maintenance of Golgi apparatus integrity.     

Recruitment of the actin-binding protein HIP-55 to the immunological synapse regulates T cell receptor signaling and endocytosis

Actin cytoskeleton dynamics critically regulate T cell activation. We found that the cytoplasmic adaptor HIP-55, a Src/Syk-kinases substrate and member of the drebrin/Abp1 family of actin-binding proteins, localized to the T cell-antigen-presenting cell (APC) contact site in an antigen-dependent manner. Using green fluorescent protein fusion proteins, both Src homology 3 (SH3) and actin binding domains were found necessary for recruitment at the T cell-APC interface. HIP-55 was not implicated in conjugate formation and actin polymerization but regulated distal signaling events through binding and activation of hematopoietic progenitor kinase 1 (HPK1), a germinal center kinase (GCK) family kinase involved in negative signaling in T cells. Using RNA interference and overexpression experiments, the HIP-55-HPK1 complex was found to negatively regulate nuclear factor of activated T cell (NFAT) activation by the T cell antigen receptor. Moreover, we show that HIP-55, which partly co-localized with early endocytic compartments, promoted both basal and ligand-dependent T cell receptor (TCR) down-modulation, resulting in a decreased TCR expression. SH3 and actin-depolymerizing factor homology domains were required for this function. As controls, the expression of CD28 and the glycosylphosphatidylinositol-linked protein CD59 was not affected by HIP-55 overexpression. These results suggest that, in addition to binding to HPK1, HIP-55 might negatively regulate TCR signaling through down-regulation of TCR expression. Our findings show that HIP-55 is a key novel component of the immunological synapse that modulates T cell activation by connecting actin cytoskeleton and TCRs to gene activation and endocytic processes.     

HS 1-associated protein X-1 is cleaved by caspase-3 during apoptosis

Caspase-3 (CASP3) plays a key role in apoptosis. In this study, HAX-1 was identified as a new substrate of CASP3 during apoptosis. HAX-1 was cleaved by CASP3 during etoposide-(ETO) induced apoptosis, and this event was inhibited by a CASP3-specific inhibitor. The cleavage site of HAX-1, at Asp(127), was located using N-terminal amino acid sequencing of in vitro cleavage products of recombinant HAX-1. Overexpression of HAX-1 inhibited ETO-induced apoptotic cell death. It also inhibited CASP3 activity. Together, these results suggest that HAX-1, a substrate of CASP3, inhibits the apoptotic process by inhibiting CASP3 activity.     

Abl-SH3 binding protein 2, 3BP2, interacts with CIN85 and HIP-55

The adapter 3BP2 is involved in leukocyte signaling downstream Src/Syk-kinases coupled immunoreceptors. Here, we show that 3BP2 directly interacts with the endocytic scaffold protein CIN85 and the actin-binding protein HIP-55. 3BP2 co-localized with CIN85 and HIP-55 in T cell rafts and at the T cell/APC synapse, an active zone of receptors and proteins recycling. A binding region of CIN85 SH3 domains on 3BP2 was mapped to a PVPTPR motif in the first proline-rich region of 3BP2, whereas the C-terminal SH3 domain of HIP-55 bound a more distal proline-rich domain of 3BP2. Together, our data suggest an unexpected role of 3BP2 in endocytic and cytoskeletal regulation through its interaction with CIN85 and HIP-55.     

The SH3 domain of HS1 protein recognizes lysine-rich polyproline motifs

HS1 is a protein involved in erythroid proliferation and apoptotic cell death, containing several structurally significant motifs including a C-terminal SH3 domain. HPK1 is a member of the Ste20-related kinase family, which contains four proline-rich sequences and is constitutively associated with HS1 in hematopoietic cells. Recombinant fusion protein GST-SH3_HS1 was expressed to assess the binding properties of 16 peptides derived from the HPK1 proline-rich regions. The binding affinities were determined by non-immobilized ligand interaction assay by circular dichroism. Our results revealed that the classical PxxPxK class II binding motif is not sufficient to induce the interaction with the GST-SH3_HS1 domain, an event dependent on the presence of additional basic residue(s) located at the C-terminus of the PxxPxK motif: Lys₋₅ in P2 peptide and Lys₋₈ in P4c peptide. Lys replacement by Arg residues decreases the ligand binding affinity. The finding that both SH3_HS1 domain and full-length HS1 protein bind to P2 peptide with similar affinity demonstrates that the whole protein sequence does not affect the interaction properties of the domain. In silico models of SH3_HS1 as a complex with P2 or P4c highlight the domain residues that interact with the recognition determinants of the peptide ligand and that cooperate in the complex stabilization.     

A novel src homology 3 domain-containing adaptor protein, HIP-55, that interacts with hematopoietic progenitor kinase 1

Hematopoietic progenitor kinase 1 (HPK1) is a member of the mitogen-activated protein kinase kinase kinase kinase (MAP4K) family and an upstream activator of the c-Jun N-terminal kinase (JNK) signaling cascade. HPK1 interacts, through its proline-rich domains, with growth factor receptor-bound 2 (Grb2), CT10-regulated kinase (Crk), and Crk-like (CrkL) adaptor proteins. We identified a novel HPK1-interacting protein of 55 kDa (HIP-55), similar to the mouse SH3P7 protein, containing an N-terminal actin-binding domain and a C-terminal Src homology 3 domain. We found that HPK1 bound to HIP-55 both in vitro and in vivo. When co-transfected, HIP-55 increased HPK1's kinase activity as well as JNK1's kinase activity. A dominant-negative HPK1 mutant blocked activation of JNK1 by HIP-55 showing that HIP-55 activates the JNK1 signaling pathway via HPK1. Our results identify a novel protein, HIP-55, that binds to HPK1 and regulates the JNK1 signaling cascade.     

Platelet-derived growth factor-dependent activation of phosphatidylinositol 3-kinase is regulated by receptor binding of SH2-domain-containing proteins which influence Ras activity.

Upon binding of platelet-derived growth factor (PDGF), the PDGF beta receptor (PDGFR) undergoes autophosphorylation on distinct tyrosine residues and binds several SH2-domain-containing signal relay enzymes, including phosphatidylinositol 3-kinase (PI3K), phospholipase C gamma (PLC gamma), the GTPase-activating protein of Ras (RasGAP), and the tyrosine phosphatase SHP-2. In this study, we have investigated whether PDGF-dependent PI3K activation is affected by the other proteins that associate with the PDGFR. We constructed and characterized a series of PDGFR mutants which contain binding sites for PI3K as well as one additional protein, either RasGAP, SHP-2, or PLC gamma. While all of the receptors had wild-type levels of PDGF-stimulated tyrosine kinase activity and associated with comparable amounts of PI3K activity, their abilities to trigger accumulation of PI3K products in vivo differed dramatically. The wild-type receptor, as well as receptors that recruited PI3K or PI3K and SHP-2, were all capable of fully activating PI3K. In contrast, receptors that associated with PI3K and RasGAP or PI3K and PLC gamma displayed a greatly reduced ability to stimulate production of PI3K products. When this series of receptors was tested for their ability to activate Ras, we observed a strong positive correlation between Ras activation and PI3K activation. Further investigation of the relationship between Ras and PI3K indicated that Ras was upstream of PI3K. Thus, activation of PI3K requires not only binding of PI3K to the tyrosine-phosphorylated PDGFR but accumulation of GTP-bound Ras as well. Furthermore, PLC gamma and RasGAP negatively modulate PDGF-dependent PI3K activation. Finally, PDGF-stimulated signal relay can be regulated by altering the ratio of SH2-domain-containing enzymes that are recruited to the PDGFR.     

Caspase-mediated cleavage of Beclin-1 inactivates Beclin-1-induced autophagy and enhances apoptosis by promoting the release of proapoptotic factors from mitochondria

Autophagy and apoptosis are two important and interconnected stress-response mechanisms. However, the molecular interplay between these two pathways is not fully understood. To study the fate and function of autophagic proteins at the onset of apoptosis, we used a cellular model system in which autophagy precedes apoptosis. IL-3 depletion of Ba/F3 cells caused caspase (casp)-mediated cleavage of Beclin-1 and PI3KC3, two crucial components of the autophagy-inducing complex. We identified two casp cleavage sites in Beclin-1, TDVD¹³³ and DQLD¹⁴⁹, cleavage at which yields fragments lacking the autophagy-inducing capacity. Noteworthy, the C-terminal fragment, Beclin-1-C, localized predominantly at the mitochondria and sensitized the cells to apoptosis. Moreover, on isolated mitochondria, recombinant Beclin-1-C was able to induce the release of proapoptotic factors. These findings point to a mechanism by which casp-dependent generation of Beclin-1-C creates an amplifying loop enhancing apoptosis upon growth factor withdrawal.     

Characterization of beta-N-acetylglucosaminidase cleavage by caspase-3 during apoptosis

Beta-O-linked N-acetylglucosamine is a dynamic post-translational modification involved in protein regulation in a manner similar to phosphorylation. Removal of N-acetylglucosamine is regulated by beta-N-acetylglucosaminidase (O-GlcNAcase), which was previously shown to be a substrate of caspase-3 in vitro. Here we show that O-GlcNAcase is cleaved by caspase-3 into two fragments during apoptosis, an N-terminal fragment containing the O-GlcNAcase active site and a C-terminal fragment containing a region with homology to GCN5 histone acetyl-transferases. The caspase-3 cleavage site of O-GlcNAcase, mapped by Edman sequencing, is a noncanonical recognition site that occurs after Asp-413 of the SVVD sequence in human O-GlcNAcase. A point mutation, D413A, abrogates cleavage by caspase-3 both in vitro and in vivo. Finally, we show that O-GlcNAcase activity is not affected by caspase-3 cleavage because the N- and C-terminal O-GlcNAcase fragments remain associated after the cleavage. Furthermore, when co-expressed simultaneously in the same cell, the N-terminal and C-terminal caspase fragments associate to reconstitute O-GlcNAcase enzymatic activity. These studies support the identification of O-GlcNAcase as a caspase-3 substrate with a novel caspase-3 cleavage site and provide insight about O-GlcNAcase regulation during apoptosis.     

Interaction of the metalloprotease disintegrins MDC9 and MDC15 with two SH3 domain-containing proteins, endophilin I and SH3PX1.

Metalloprotease disintegrins (a disintegrin and metalloprotease (ADAM) and metalloprotease, disintegrin, cysteine-rich proteins (MDC)) are a family of membrane-anchored glycoproteins that function in diverse biological processes, including fertilization, neurogenesis, myogenesis, and ectodomain processing of cytokines and other proteins. The cytoplasmic domains of ADAMs often include putative signaling motifs, such as proline-rich SH3 ligand domains, suggesting that interactions with cytoplasmic proteins may affect metalloprotease disintegrin function. Here we report that two SH3 domain-containing proteins, endophilin I (SH3GL2, SH3p4) and a novel SH3 domain- and phox homology (PX) domain-containing protein, termed SH3PX1, can interact with the cytoplasmic domains of the metalloprotease disintegrins MDC9 and MDC15. These interactions were initially identified in a yeast two-hybrid screen and then confirmed using bacterial fusion proteins and co-immunoprecipitations from eukaryotic cells expressing both binding partners. SH3PX1 and endophilin I both preferentially bind the precursor but not the processed form of MDC9 and MDC15 in COS-7 cells. Since rat endophilin I is thought to play a role in synaptic vesicle endocytosis and SH3PX1 has sequence similarity to sorting nexins in yeast, we propose that endophilin I and SH3PX1 may have a role in regulating the function of MDC9 and MDC15 by influencing their intracellular processing, transport, or final subcellular localization.     

Identification of Src, Fyn, and Lyn SH3-binding proteins: implications for a function of SH3 domains.

Src homology 3 (SH3) domains mediate protein-protein interactions necessary for the coupling of cellular proteins involved in intracellular signal transduction. We previously established solution-binding conditions that allow affinity isolation of Src SH3-binding proteins from cellular extracts (Z. Weng, J. A. Taylor, C. E. Turner, J. S. Brugge, and C. Seidel-Dugan, J. Biol. Chem. 268:14956-14963, 1993). In this report, we identified three of these proteins: Shc, a signaling protein that couples membrane tyrosine kinases with Ras; p62, a protein which can bind to p21rasGAP; and heterogeneous nuclear ribonucleoprotein K, a pre-mRNA-binding protein. All of these proteins contain proline-rich peptide motifs that could serve as SH3 domain ligands, and the binding of these proteins to the Src SH3 domain was inhibited with a proline-rich Src SH3 peptide ligand. These three proteins, as well as most of the other Src SH3 ligands, also bound to the SH3 domains of the closely related protein tyrosine kinases Fyn and Lyn. However, Src- and Lyn-specific SH3-binding proteins were also detected, suggesting subtle differences in the binding specificity of the SH3 domains from these related proteins. Several Src SH3-binding proteins were phosphorylated in Src-transformed cells. The phosphorylation of these proteins was not detected in cells transformed by a mutant variant of Src lacking the SH3 domain, while there was little change in tyrosine phosphorylation of other Src-induced phosphoproteins. In addition, the coprecipitation of v-Src with two tyrosyl-phosphorylated proteins with M(r)s of 62,000 and 130,000 was inhibited by incubation with a Src SH3 peptide ligand, suggesting that the binding of these substrate proteins is dependent on interactions with the SH3 domain. These results strongly suggest a role for the Src SH3 domain in the recruitment of substrates to this protein tyrosine kinase, either through direct interaction with the SH3 domain or indirectly through interactions with proteins that bind to the SH3 domain.     

Caspase-mediated cleavage and DNase activity of the translation initiation factor 3, subunit G (eIF3g)

Eukaryotic translation initiation factor 3 is composed of 13 subunits (eIF3a through eIF3m) and plays an essential role in translation. During apoptosis, several caspases rapidly down-regulate protein synthesis by cleaving eIF4G, -4B, -3j, and -2α. In this study, we found that the activation of caspases by cisplatin in T24 cells induces the cleavage of subunit G of the eIF3 complex (eIF3g). The cleavage site (SLRD²²⁰G) was identified, and we found that the cleaved N-terminus was translocated to the nucleus, activating caspase-3, and that it also showed a strong DNase activity. These data demonstrate the important roles of eIF3g in the translation initiation machinery and in DNA degradation during apoptosis.     

Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis

During apoptosis, the permeabilization of the mitochondrial outer membrane allows the release of cytochrome c, which induces caspase activation to orchestrate the death of the cell. Mitochondria rapidly lose their transmembrane potential (Delta Psi m) and generate reactive oxygen species (ROS), both of which are likely to contribute to the dismantling of the cell. Here we show that both the rapid loss of Delta Psi m and the generation of ROS are due to the effects of activated caspases on mitochondrial electron transport complexes I and II. Caspase-3 disrupts oxygen consumption induced by complex I and II substrates but not that induced by electron transfer to complex IV. Similarly, Delta Psi m generated in the presence of complex I or II substrates is disrupted by caspase-3, and ROS are produced. Complex III activity measured by cytochrome c reduction remains intact after caspase-3 treatment. In apoptotic cells, electron transport and oxygen consumption that depends on complex I or II was disrupted in a caspase-dependent manner. Our results indicate that after cytochrome c release the activation of caspases feeds back on the permeabilized mitochondria to damage mitochondrial function (loss of Delta Psi m) and generate ROS through effects of caspases on complex I and II in the electron transport chain.     

Proteolytic cleavage of phospholipase C-gamma1 during apoptosis in Molt-4 cells.

Apoptosis is a cell suicide mechanism that requires the activation of cellular death proteases for its induction. We examined whether the progress of apoptosis involves cleavage of phospholipase C-gamma1 (PLC-gamma1), which plays a pivotal role in mitogenic signaling pathway. Pretreatment of T leukemic Molt-4 cells with PLC inhibitors such as U-73122 or ET-18-OCH(3) potentiated etoposide-induced apoptosis in these cells. PLC-gamma1 was fragmented when Molt-4 cells were treated with several apoptotic stimuli such as etoposide, ceramides, and tumor necrosis factor alpha. Cleavage of PLC-gamma1 was blocked by overexpression of Bcl-2 and by specific inhibitors of caspases such as Z-DEVD-CH(2)F and YVAD-cmk. Purified caspase-3 and caspase-7, group II caspases, cleaved PLC-gamma1 in vitro and generated a cleavage product of the same size as that observed in vivo, suggesting that PLC-gamma1 is cleaved by group II caspases in vivo. From point mutagenesis studies, Ala-Glu-Pro-Asp(770) was identified to be a cleavage site within PLC-gamma1. Epidermal growth factor receptor (EGFR) -induced tyrosine phosphorylation of PLC-gamma1 resulted in resistance to cleavage by caspase-3 in vitro. Furthermore, cleaved PLC-gamma1 could not be tyrosine-phosphorylated by EGFR in vitro. In addition, tyrosine-phosphorylated PLC-gamma1 was not significantly cleaved during etoposide-induced apoptosis in Molt-4 cells. This suggests that the growth factor-induced tyrosine phosphorylation may suppress apoptosis-induced fragmentation of PLC-gamma1. We provide evidence for the biochemical relationship between PLC-gamma1-mediated signal pathway and apoptotic signal pathway, indicating that the defect of PLC-gamma1-mediated signaling pathway can facilitate an apoptotic progression.