A role for the cleaved cytoplasmic domain of E-cadherin in the nucleus

Cell-cell contacts play a vital role in intracellular signaling, although the molecular mechanisms of these signaling pathways are not fully understood. E-cadherin, an important mediator of cell-cell adhesions, has been shown to be cleaved by gamma-secretase. This cleavage releases a fragment of E-cadherin, E-cadherin C-terminal fragment 2 (E-cad/CTF2), into the cytosol. Here, we study the fate and function of this fragment. First, we show that coexpression of the cadherin-binding protein, p120 catenin (p120), enhances the nuclear translocation of E-cad/CTF2. By knocking down p120 with short interfering RNA, we also demonstrate that p120 is necessary for the nuclear localization of E-cad/CTF2. Furthermore, p120 enhances and is required for the specific binding of E-cad/CTF2 to DNA. Finally, we show that E-cad/CTF2 can regulate the p120-Kaiso-mediated signaling pathway in the nucleus. These data indicate a novel role for cleaved E-cadherin in the nucleus.     

C-terminal Src Kinase (Csk)-mediated Phosphorylation of Eukaryotic Elongation Factor 2 (eEF2) Promotes Proteolytic Cleavage and Nuclear Translocation of eEF2

Protein-tyrosine kinase C-terminal Src kinase (Csk) was originally purified as a kinase for phosphorylating Src and other Src family kinases. The phosphorylation of a C-terminal tyrosine residue of Src family kinases suppresses their kinase activity. Therefore, most physiological studies regarding Csk function have been focused on Csk as a negative regulator of Src family tyrosine kinases and as a potential tumor suppressor. Paradoxically, the protein levels of Csk were elevated in some human carcinomas. In this report, we show that eukaryotic elongation factor 2 (eEF2) is a new protein substrate of Csk and could locate in the nucleus. We demonstrate that Csk-mediated phosphorylation of eEF2 has no effect on its cytoplasmic function in regulating protein translation. However, phosphorylation of eEF2 enhances its proteolytic cleavage and the nuclear translocation of the cleaved eEF2 through a SUMOylation-regulated process. Furthermore, we show that cleaved fragments of eEF2 can induce nuclear morphological changes and aneuploidy similar to those in cancer cells, suggesting that there is an additional mechanism for Csk in tumorigenesis through regulation of eEF2 subcellular localization.     

Caspase-dependent cleavage of c-Abl contributes to apoptosis

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.     

Induction of apoptosis is driven by nuclear retention of protein kinase C delta

Protein kinase C delta (PKC delta) mediates apoptosis downstream of many apoptotic stimuli. Because of its ubiquitous expression, tight regulation of the proapoptotic function of PKC delta is critical for cell survival. Full-length PKC delta is found in all cells, whereas the catalytic fragment of PKC delta, generated by caspase cleavage, is only present in cells undergoing apoptosis. Here we show that full-length PKC delta transiently accumulates in the nucleus in response to etoposide and that nuclear translocation precedes caspase cleavage of PKC delta. Nuclear PKC delta is either cleaved by caspase 3, resulting in accumulation of the catalytic fragment in the nucleus, or rapidly exported by a Crm1-sensitive pathway, thereby assuring that sustained nuclear accumulation of PKC delta is coupled to caspase activation. Nuclear accumulation of PKC delta is necessary for caspase cleavage, as mutants of PKC delta that do not translocate to the nucleus are not cleaved. However, caspase cleavage of PKC delta per se is not required for apoptosis, as an uncleavable form of PKC delta induces apoptosis when retained in the nucleus by the addition of an SV-40 nuclear localization signal. Finally, we show that kinase negative full-length PKC delta does not translocate to the nucleus in apoptotic cells but instead inhibits apoptosis by blocking nuclear import of endogenous PKC delta. These studies demonstrate that generation of the PKC delta catalytic fragment is a critical step for commitment to apoptosis and that nuclear import and export of PKC delta plays a key role in regulating the survival/death pathway.     

Cell type-specific cleavage of nucleocapsid protein by effector caspases during SARS coronavirus infection

The epidemic outbreak of severe acute respiratory syndrome (SARS) in 2003 was caused by a novel coronavirus (CoV), designated SARS-CoV. The RNA genome of SARS-CoV is complexed by the nucleocapsid protein (N) to form a helical nucleocapsid. Besides this primary function, N seems to be involved in apoptotic scenarios. We show that upon infection of Vero E6 cells with SARS-CoV, which elicits a pronounced cytopathic effect and a high viral titer, N is cleaved by caspases. In contrast, in SARS-CoV-infected Caco-2 cells, which show a moderate cytopathic effect and a low viral titer, this processing of N was not observed. To further verify these observations, we transiently expressed N in different cell lines. Caco-2 and N2a cells served as models for persistent SARS-CoV infection, whereas Vero E6 and A549 cells did as prototype cell lines lytically infected by SARS-CoV. The experiments revealed that N induces the intrinsic apoptotic pathway, resulting in processing of N at residues 400 and 403 by caspase-6 and/or caspase-3. Of note, caspase activation is highly cell type specific in SARS-CoV-infected as well as transiently transfected cells. In Caco-2 and N2a cells, almost no N-processing was detectable. In Vero E6 and A549 cells, a high proportion of N was cleaved by caspases. Moreover, we examined the subcellular localization of SARS-CoV N in these cell lines. In transfected Vero E6 and A549 cells, SARS-CoV N was localized both in the cytoplasm and nucleus, whereas in Caco-2 and N2a cells, nearly no nuclear localization was observed. In addition, our studies indicate that the nuclear localization of N is essential for its caspase-6-mediated cleavage. These data suggest a correlation among the replication cycle of SARS-CoV, subcellular localization of N, induction of apoptosis, and the subsequent activation of caspases leading to cleavage of N.     

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.     

PKCdelta-dependent cleavage and nuclear translocation of annexin A1 by phorbol 12-myristate 13-acetate.

Annexin A1 (ANX-1), a calcium-dependent, phospholipid binding protein, is known to be involved in diverse cellular processes, including regulation of cell growth and differentiation, apoptosis, and inflammation. The mitogen phorbol 12-myristate 13-acetate (PMA) induces expression and phosphorylation of ANX-1. However, the roles of ANX-1 in PMA-induced signal transduction is unknown. Here, we study the cellular localization of ANX-1 in the PMA-induced signal transduction process. We have found that PMA induces the cleavage of ANX-1 in human embryonic kidney (HEK) 293 cells, and that the cleaved form of ANX-1 translocates to the nucleus. The PMA-induced nuclear translocation of ANX-1 was inhibited by the protein kinase C (PKC)delta-specific inhibitor rottlerin, indicating that PKCdelta plays a role in nuclear translocation of the cleaved ANX-1. We propose a novel mechanism of PMA-induced translocation of ANX-1 to the nucleus that may participate in the regulation of cell proliferation and differentiation.     

Caspase-3-mediated cleavage of Cdc6 induces nuclear localization of p49-truncated Cdc6 and apoptosis

We show that Cdc6, an essential initiation factor for DNA replication, undergoes caspase-3-mediated cleavage in the early stages of apoptosis in HeLa cells and SK-HEP-1 cells induced by etoposide, paclitaxel, ginsenoside Rh2, or tumor necrosis factor-related apoptosis-inducing ligand. The cleavage occurs at the SEVD442/G motif and generates an N-terminal truncated Cdc6 fragment (p49-tCdc6) that lacks the carboxy-terminal nuclear export sequence. Cdc6 is known to be phosphorylated by cyclin A-cyclin dependent kinase 2 (Cdk2), an event that promotes its exit from the nucleus and probably blocks it from initiating inappropriate DNA replication. In contrast, p49-tCdc6 translocation to the cytoplasm is markedly reduced under the up-regulated conditions of Cdk2 activity, which is possibly due to the loss of nuclear export sequence. Thus, truncation of Cdc6 results in an increased nuclear retention of p49-tCdc6 that could act as a dominant negative inhibitor of DNA replication and its accumulation in the nucleus could promote apoptosis. Supporting this is that the ectopic expression of p49-tCdc6 not only promotes apoptosis of etoposide-induced HeLa cells but also induces apoptosis in untreated cells. Thus, the caspase-mediated cleavage of Cdc6 creates a truncated Cdc6 fragment that is retained in the nucleus and induces apoptosis.     

Identification of a novel protein complex containing annexin VI, Fyn, Pyk2, and the p120(GAP) C2 domain

p120(GAP) (RasGAP) has been proposed to function as both an inhibitor and effector of Ras. Previously we have shown that RasGAP contains a C2 domain which mediates both Ca(2+)-dependent membrane association and protein-protein interactions. Specifically, three proteins have been isolated in a complex with the C2 domain of RasGAP; these are the Ca(2+)-dependent lipid binding protein annexin VI (p70) and two previously unidentified proteins, p55 and p120. Here we provide evidence that p55 is the Src family kinase Fyn and p120 is the focal adhesion kinase family member Pyk2. In addition, in vitro binding assays indicate that Fyn, but not Pyk2 binds directly to annexin VI. Finally, co-immunoprecipitation studies in Rat-1 fibroblasts confirm that Fyn, Pyk2, annexin VI and RasGAP can form a protein complex in mammalian cells.     

Specific phosphorylation of p120-catenin regulatory domain differently modulates its binding to RhoA

p120-catenin is an adherens junction-associated protein that controls E-cadherin function and stability. p120-catenin also binds intracellular proteins, such as the small GTPase RhoA. In this paper, we identify the p120-catenin N-terminal regulatory domain as the docking site for RhoA. Moreover, we demonstrate that the binding of RhoA to p120-catenin is tightly controlled by the Src family-dependent phosphorylation of p120-catenin on tyrosine residues. The phosphorylation induced by Src and Fyn tyrosine kinases on p120-catenin induces opposite effects on RhoA binding. Fyn, by phosphorylating a residue located in the regulatory domain of p120-catenin (Tyr112), inhibits the interaction of this protein with RhoA. By contrast, the phosphorylation of Tyr217 and Tyr228 by Src promotes a better affinity of p120-catenin towards RhoA. In agreement with these biochemical data, results obtained in cell lines support the important role of these phosphorylation sites in the regulation of RhoA activity by p120-catenin. Taken together, these observations uncover a new regulatory mechanism acting on p120-catenin that contributes to the fine-tuned regulation of the RhoA pathways during specific signaling events.     

Cleavage at the carboxyl-terminus of Ku80 during apoptosis in human Jurkat T cells

We have previously reported that the amount of Apg-2, an Hsp110 family protein, decreases during apoptosis in Jurkat T cells. Since we hypothesized that Apg-2 would be cleaved by caspase-3 during apoptosis, a cleavage-site-directed antibody was raised against the carboxyl-terminus of the Apg-2 fragment that appears after the cleavage by caspase-3. Although this antibody could not detect the Apg-2 fragment in apoptotic cells, three additional fragments were unexpectedly detected. Based on the results of microsequencing, one of these fragments was identified as Ku80. Ku80 is a nuclear protein and a component of DNA-dependent protein kinase (DNA-PK). In this study, we observed that Ku80 is cleaved at Asp-730 residue during apoptosis, and this cleavage occurs in the nucleus in the early apoptotic phase. Furthermore, Ku80 is distributed in the cytoplasm of nuclear fragmented apoptotic cells, although the cleaved fragment contains the nuclear-localization signal (NLS). Our study clearly shows that Ku80 is cleaved in the nucleus, and distributes in the cytoplasm during apoptosis.