Daniela Barilà

Src kinase phosphorylates Caspase-8 on Tyr380: a novel mechanism of apoptosis suppression

We identified Caspase‐8 as a new substrate for Src kinase. Phosphorylation occurs on Tyr380, situated in the linker region between the large and the small subunits of human Procaspase‐8, and results in downregulation of Caspase‐8 proapoptotic function. Src activation triggers Caspase‐8 phosphorylation on Tyr380 and impairs Fas‐induced apoptosis. Accordingly, Src failed to protect Caspase‐8‐defective human cells in which a Caspase‐8‐Y380F mutant is expressed from Fas‐induced cell death. Remarkably, Src activation upon EGF‐receptor stimulation triggers endogenous Caspase‐8 phosphorylation and prevents Fas‐induced apoptosis. Tyr380 is phosphorylated also in human colon cancers where Src is aberrantly activated. These data provide the first evidence for a direct role of tyrosine phosphorylation in the control of caspases and reveal a new mechanism through which tyrosine kinases inhibit apoptosis and participate in tumor progression.

c-Abl is an effector of Src for growth factor-induced c-myc expression and DNA synthesis

The mechanism by which the ubiquitously expressed Src family kinases regulate mitogenesis is not well understood. Here we report that cytoplasmic tyrosine kinase c‐Abl is an important effector of c‐Src for PDGF‐ and serum‐induced DNA synthesis. Inactivation of cytoplasmic c‐Abl by the kinase‐ inactive Abl‐PP‐K− (AblP242E/P249E/K290M) or by microinjection of Abl neutralizing antibodies inhibited mitogenesis. The kinase‐inactive SrcK295M induced a G1 block that was overcome by the constitutively active Abl‐PP (AblP242E/P249E). Conversely, the inhibitory effect of Abl‐PP‐K− was not compensated by Src. c‐Src‐induced c‐Abl activation involves phosphorylation of Y245 and Y412, two residues required for c‐Abl mitogenic function. Finally, we found that p53 inactivation and c‐myc expression, two cell cycle events regulated by Src during mitogenesis, also implied c‐Abl: c‐Abl function was dispensable in cells deficient in active p53 and inhibition of c‐Abl reduced mitogen‐induced c‐myc expression. These data identify a novel function of cytoplasmic c‐Abl in the signalling pathways regulating growth factor‐induced c‐myc expression and we propose the existence of a tyro sine kinase signalling cascade (PDGFR/c‐Src/c‐Abl) important for mitogenesis.

A nuclear tyrosine phosphorylation circuit: c-Jun as an activator and substrate of c-Abl and JNK

The nuclear function of the c‐Abl tyrosine kinase is not well understood. In order to identify nuclear substrates of Abl, we constructed a constitutively active and nuclear form of the protein. We found that active nuclear Abl efficiently phosphorylate c‐Jun, a transcription factor not previously known to be tyrosine phosphorylated. After phosphorylation of c‐Jun by Abl on Tyr170, both proteins interacted via the SH2 domain of Abl. Surprisingly, elevated levels of c‐Jun activated nuclear Abl, resulting in activation of the JNK serine/threonine kinase. This phosphorylation circuit generates nuclear tyrosine phosphorylation and represents a reversal of previously known signalling models.

Caspase-8 association with the focal adhesion complex promotes tumor cell migration and metastasis.

Caspase-8 is a proapoptotic protease that suppresses neuroblastoma metastasis by inducing programmed cell death. Paradoxically, caspase-8 can also promote cell migration among nonapoptotic cells; here, we show that caspase-8 can promote metastasis when apoptosis is compromised. Migration is enhanced by caspase-8 recruitment to the cellular migration machinery following integrin ligation. Caspase-8 catalytic activity is not required for caspase-8-enhanced cell migration; rather, caspase-8 interacts with a multiprotein complex that can include focal adhesion kinase and calpain 2 (CPN2), enhancing cleavage of focal adhesion substrates and cell migration. Caspase-8 association with CPN2/calpastatin disrupts calpastatin-mediated inhibition of CPN2. In vivo, knockdown of either caspase-8 or CPN2 disrupts metastasis among apoptosis-resistant tumors. This unexpected molecular collaboration provides an explanation for the continued or elevated expression of caspase-8 observed in many tumors.

The Dri 42 gene, whose expression is up-regulated during epithelial differentiation, encodes a novel endoplasmic reticulum resident transmembrane protein

A search for novel genes that are up-regulated during development and differentiation of the epithelial cells of the intestinal mucosa led us to the isolation of the Dri 42 cDNA clone (Dri, differentially expressed in rat intestine). The nucleotide sequence of the full-length cDNA has shown that it encodes a 35.5-kDa protein with one consensus sequence for N-linked glycosylation and alternating hydrophilic and hydrophobic domains. To determine the intracellular localization of Dri 42 we have raised polyclonal antibodies in hens against a bacterially produced Dri 42-glutathione S-transferase fusion protein. Immunofluorescence detection with these antibodies has shown specific staining of the endoplasmic reticulum (ER) in the relatively undifferentiated fetal rat intestinal cell line FRIC B and in sections of rat small intestine. ER membrane localization of Dri 42 was confirmed by laser confocal microscopy of polarized Madin-Darby canine kidney cells overexpressing a Dri 42-chloramphenicol acetyltransferase (CAT) fusion protein by transfection. Pulse labeling experiments on transiently transfected cells demonstrated that the protein does not acquire Golgi modifications up to 4 h after synthesis, thus indicating that Dri 42 is an ER resident protein. The transmembrane disposition of Dri 42 was studied using in vitro insertion of Dri 42-CAT fusion proteins into microsomal membranes. The fusion proteins consisted of several different lengths of truncated Dri 42 and a reporter protein, CAT, that was linked in-frame after each hydrophobic segment. We found that hydrophobic segments H1, H3, and H5 had a signal/anchor function, and that membrane insertion of Dri 42 was achieved co-translationally by the action of a series of alternating insertion signals and halt transfer signals, resulting in the exposure of both termini of the protein to the cytosolic side. The functional implications of the structure and localization of Dri 42, whose primary sequence does not share significant homology to any previously described protein, are discussed.

Caspase-Dependent Cleavage of c-Abl Contributes to Apoptosis

ABSTRACT The nonreceptor tyrosine kinase c-Abl may contribute to the regulation of apoptosis. c-Abl activity is induced in the nucleus upon DNA damage, and its activation is required for execution of the apoptotic program. Recently, activation of nuclear c-Abl during death receptor-induced apoptosis has been reported; however, the mechanism remains largely obscure. Here we show that c-Abl is cleaved by caspases during tumor necrosis factor- and Fas receptor-induced apoptosis. Cleavage at the very C-terminal region of c-Abl occurs mainly in the cytoplasmic compartment and generates a 120-kDa fragment that lacks the nuclear export signal and the actin-binding region but retains the intact kinase domain, the three nuclear localization signals, and the DNA-binding domain. Upon caspase cleavage, the 120-kDa fragment accumulates in the nucleus. Transient-transfection experiments show that cleavage of c-Abl may affect the efficiency of Fas-induced cell death. These data reveal a novel mechanism by which caspases can recruit c-Abl to the nuclear compartment and to the mammalian apoptotic program.

Identification of a Critical Tyrosine Residue in Caspase 8 That Promotes Cell Migration

Caspase 8 is a critical upstream initiator of programmed cell death but, paradoxically, has also been shown to promote cell migration. Here, we show that tyrosine 380 in the linker loop of human caspase 8 is a critical switch determining caspase 8 function. Our studies show that, in addition to its cytosolic distribution, caspase 8 is recruited to lamella of migrating cells. Although the catalytic domain of caspase 8 is sufficient for recruitment and promotion of cell migration, catalytic activity per se is not required. Instead, we find that integrin-mediated adhesion promotes caspase 8 phosphorylation on tyrosine 380. Accordingly, mutation of this site compromises localization to the periphery and the potentiation of cell migration. Mechanistically, this linker region of caspase 8 acts as a Src homology 2 binding site. In particular, tyrosine 380 is critical for interaction with Src homology 2 domains. The results identify a novel mechanism by which caspase 8 is recruited to the lamella of a migrating cell, promoting cell migration independent of its protease activity.

ATM-depletion in breast cancer cells confers sensitivity to PARP inhibition.

BackgroundMutations in the DNA damage response (DDR) factors, breast cancer 1 (BRCA1) and BRCA2, sensitize tumor cells to poly(ADP-ribose) polymerase (PARP) inhibitors. The ataxia telangiectasia mutated (ATM) kinase is a key DDR protein whose heterozygous germline mutation is a moderate–risk factor for developing breast cancer. In this study, we examined whether ATM inactivation in breast cancer cell lines confers sensitivity to PARP inhibitors.MethodsWild-type BRCA1/2 breast cancer cells (i.e., MCF-7 and ZR-75-1 lines) were genetically manipulated to downregulate ATM expression then assayed for cytostaticity/cytotoxicity upon treatment with PARP inhibitors, olaparib and iniparib.ResultsWhen ATM-depleted cells and their relative controls were treated with olaparib (a competitive PARP-1/2 inhibitor) and iniparib (a molecule originally described as a covalent PARP-1 inhibitor) a different response to the two compounds was observed. ATM-depletion sensitized both MCF-7 and ZR-75-1 cells to olaparib-treatment, as assessed by short and long survival assays and cell cycle profiles. In contrast, iniparib induced only a mild, ATM-dependent cytostatic effect in MCF-7 cells whereas ZR-75-1 cells were sensitive to this drug, independently of ATM inactivation. These latest results might be explained by recent observations indicating that iniparib acts with mechanisms other than PARP inhibition.ConclusionsThese data indicate that ATM-depletion can sensitize breast cancer cells to PARP inhibition, suggesting a potential in the treatment of breast cancers low in ATM protein expression/activity, such as those arising in mutant ATM heterozygous carriers.

Up-Regulation of c-Jun Inhibits Proliferation and Induces Apoptosis via Caspase-Triggered c-Abl Cleavage in Human Multiple Myeloma

Here we show the antimyeloma cytotoxicity of adaphostin and carried out expression profiling of adaphostin-treated multiple myeloma (MM) cells to identify its molecular targets. Surprisingly, c-Jun was the most up-regulated gene even at the earliest point of analysis (2 h). We also observed adaphostin-induced c-Abl cleavage in immunoblot analysis. Proteasome inhibitor bortezomib, but not melphalan or dexamethasone, induced similar effects, indicating unique agent-dependent mechanisms. Using caspase inhibitors, as well as caspase-resistant mutants of c-Abl (TM-c-Abl and D565A-Abl), we then showed that c-Abl cleavage in MM cells requires caspase activity. Importantly, both overexpression of the c-Abl fragment or c-Jun and knockdown of c-Abl and c-Jun expression by small interfering RNA confirmed that adaphostin-induced c-Jun up-regulation triggers downstream caspase-mediated c-Abl cleavage, inhibition of MM cell growth, and induction of apoptosis. Finally, our data suggest that this mechanism may not only be restricted to MM but may also be important in a broad range of malignancies including erythroleukemia and solid tumors.

c‐Abl acetylation by histone acetyltransferases regulates its nuclear–cytoplasmic localization

c‐Abl function is strictly dependent on its subcellular localization. Using an in vitro approach, we identify c‐Abl as a new substrate for p300, CBP (CREB‐binding protein) and PCAF (p300/CBP‐associated factor) histone acetyltransferases. Remarkably, acetylation markedly alters its subcellular localization. Point mutagenesis indicated that Lys 730, located in the second nuclear localization signal, is the main target of p300 activity. It has previously been reported that c‐Abl accumulates in the cytoplasm during myogenic differentiation. Here, we show that c‐Abl protein is acetylated at early stages of myogenic differentiation. Indeed, acetylation on Lys 730 drives c‐Abl accumulation in the cytoplasm and promotes differentiation. Thus, Lys 730 acetylation is a novel post‐translational modification of c‐Abl and a novel mechanism for modulating its subcellular localization that contributes to myogenic differentiation.