Complete deletion of Ectromelia virus p28 impairs virus genome replication in a mouse strain, cell type, and multiplicity of infection-dependent manner.

p28 is a poxvirus-encoded E3 ubiquitin ligase that possesses an N-terminal KilA-N domain and a C-terminal RING domain. In Ectromelia virus (ECTV), disruption of the p28 RING domain severely attenuated virulence in A strain mice, which normally succumb to ECTV infection. Moreover, this mutant virus exhibited dramatically reduced genome replication and impaired factory formation in A strain mice peritoneal macrophages (PMs) infected at high multiplicity of infection (MOI) These defects were not observed in PMs isolated from C57BL/6 mice which survive ECTV infection, demonstrating that p28 functions in a context-specific manner. To further investigate p28 function, we completely deleted the p28 gene from ECTV (ECTV-Δp28). In contrast to previous findings, we found that the ECTV-Δp28 virus exhibited severely compromised virus production and genome replication in PMs isolated from A strain mice only when infected at low MOI. This defect was minimal in bone marrow-derived macrophages and two cell lines derived from A strain mice. Furthermore, this low MOI defect in virus production was also observed in PMs isolated from the susceptible BALB/c mouse strain, but not PMs isolated from C57BL/6 mice. Taken together, our data demonstrate that the requirement for ECTV p28 to establish a productive infection depends on the MOI, the cell type, as well as the mouse strain.

Poxviral ANKR/F-box Proteins: Substrate Adapters for Ubiquitylation and More.

Poxviruses are double-stranded DNA viruses that infect insects and a variety of vertebrate species. The large genomes of poxviruses contain numerous genes that allow these viruses to successfully establish infection, including those that help evade the host immune response and prevent cell death. Ankyrin-repeat (ANKR)/F-box proteins are almost exclusively found in poxviruses, and they function as substrate adapters for Skp1-Cullin-1-F-box protein (SCF) multi-subunit E3 ubiquitin (Ub)-ligases. In this regard, they use their C-terminal F-box domain to bind Skp1, Cullin-1, and Roc1 to recruit cellular E2 enzymes to facilitate the ubiquitylation, and subsequent proteasomal degradation, of proteins bound to their N-terminal ANKRs. However, these proteins do not just function as substrate adapters as they also have Ub-independent activities. In this review, we examine both Ub-dependent and -independent activities of ANKR/F-box proteins and discuss how poxviruses use these proteins to counteract the host innate immune response, uncoat their genome, replicate, block cell death, and influence transcription. Finally, we consider important outstanding questions that need to be answered in order to better understand the function of this versatile protein family.

AP-1 family transcription factors: a diverse family of proteins that regulate varied cellular activities in classical hodgkin lymphoma and ALK+ ALCL.

Classical Hodgkin lymphoma (cHL) and anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma (ALK+ ALCL) are B and T cell lymphomas respectively, which express the tumour necrosis factor receptor superfamily member, CD30. Another feature shared by cHL and ALK+ ALCL is the aberrant expression of multiple members of the activator protein-1 (AP-1) family of transcription factors which includes proteins of the Jun, Fos, ATF, and Maf subfamilies. In this review, we highlight the varied roles these proteins play in the pathobiology of these lymphomas including promoting proliferation, suppressing apoptosis, and evading the host immune response. In addition, we discuss factors contributing to the elevated expression of these transcription factors in cHL and ALK+ ALCL. Finally, we examine therapeutic strategies for these lymphomas that exploit AP-1 transcriptional targets or the signalling pathways they regulate.

The c-Jun and JunB transcription factors facilitate the transit of classical Hodgkin lymphoma tumour cells through G1.

Classical Hodgkin Lymphoma (cHL) is primarily a B cell lymphoid neoplasm and a member of the CD30-positive lymphomas. cHL and the other CD30-positive lymphomas are characterized by the elevated expression and/or constitutive activation of the activator protein-1 (AP-1) family transcription factors, c-Jun and JunB; however, the specific roles they play in the pathobiology of cHL are unclear. In this report we show that reducing either c-Jun or JunB expression with short-hairpin RNAs (shRNAs) reduced the growth of cHL cell lines in vitro and in vivo, primarily through impairing cell cycle transition through G1. We further investigated the effect of c-Jun and JunB knock-down on proliferation in another CD30-positive lymphoma, anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma (ALK+ ALCL). We found that JunB knock-down in most ALK+ ALCL cell lines examined also resulted in reduced proliferation that was associated with a G0/G1 cell cycle defect. In contrast, c-Jun knock-down in multiple ALK+ ALCL cell lines had no effect on proliferation. In summary, this study directly establishes that both c-Jun and JunB play roles in promoting HRS cell proliferation. Furthermore, we demonstrate there are similarities and differences in c-Jun and JunB function between cHL and ALK+ ALCL.

Expression of granzyme B sensitizes ALK+ ALCL tumour cells to apoptosis-inducing drugs.

BACKGROUND: The serine protease Granzyme B (GzB) is primarily expressed by cytotoxic T lymphocytes and natural killer cells, and functions in allowing these cells to induce apoptosis in virally-infected or transformed cells. Cancers of both lymphoid and non-lymphoid origin also express GzB, and in some cases this expression has been linked to pathogenesis or sensitizing tumour cells to cell death. For example, GzB expression in urothelial carcinoma was implicated in promoting tumour cell invasion, whereas its expression in nasal-type NK/T lymphomas was found to correlate with increased apoptosis. GzB expression is also a hallmark of the non-Hodgkin lymphoma, anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma (ALK+ ALCL). Given the fact that ALK+ ALCL exhibits high levels of apoptosis and is typically responsive to conventional chemotherapy, we examined whether GzB expression might play a role in sensitizing ALK+ ALCL tumour cells to apoptosis. METHODS: ALK+ ALCL cell lines stably expressing GzB or non-targeting (control) shRNA were generated and apoptosis was examined by anti-PARP western blotting and terminal deoxynucleotidyl transferase dUTP nick end labelling. Both spontaneous apoptosis and apoptosis in response to treatment with staurosporine or doxorubicin were investigated. In order to assess whether additional granzymes might be important in promoting cell death in ALK+ ALCL, we examined whether other human granzymes were expressed in ALK+ ALCL cell lines using reverse-transcriptase PCR and western blotting. RESULTS: Expression of several GzB shRNAs in multiple ALK+ ALCL cell lines resulted in a significant decrease in GzB levels and activity. While spontaneous apoptosis was similar in ALK+ ALCL cell lines expressing either GzB or control shRNA, GzB shRNA-expressing cells were less sensitive to staurosporine or doxorubicin-induced apoptosis as evidenced by reduced PARP cleavage and decreased DNA fragmentation. Furthermore, we found that GzB is the only granzyme that is expressed at significant levels in ALK+ ALCL cell lines. CONCLUSIONS: Our findings are the first to demonstrate that GzB expression sensitizes ALK+ ALCL cell lines to drug-induced apoptosis. This suggests that GzB expression may be a factor contributing to the favourable response of this lymphoma to treatment.

Cleavage of the JunB transcription factor by caspases generates a carboxyl-terminal fragment that inhibits activator protein-1 transcriptional activity.

The activator protein-1 (AP-1) family transcription factor, JunB, is an important regulator of proliferation, apoptosis, differentiation, and the immune response. In this report, we show that JunB is cleaved in a caspase-dependent manner in apoptotic anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma cell lines and that ectopically expressed JunB is cleaved in murine RAW 264.7 macrophage cells treated with the NALP1b inflammasome activator, anthrax lethal toxin. In both cases, we identify aspartic acid 137 as the caspase cleavage site and demonstrate that JunB can be directly cleaved in vitro by multiple caspases at this site. Cleavage of JunB at aspartic acid 137 separates the N-terminal transactivation domain from the C-terminal DNA binding and dimerization domains, and we show that the C-terminal cleavage fragment retains both DNA binding activity and the ability to interact with AP-1 family transcription factors. Furthermore, this fragment interferes with the binding of full-length JunB to AP-1 sites and inhibits AP-1-dependent transcription. In summary, we have identified and characterized a novel mechanism of JunB post-translational modification and demonstrate that the C-terminal JunB caspase cleavage product functions as a potent inhibitor of AP-1-dependent transcription.

The pathobiology of the oncogenic tyrosine kinase NPM-ALK: a brief update.

Extensive research has been carried out in the past two decades to study the pathobiology of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), which is an oncogenic fusion protein found exclusively in a specific type of T-cell lymphoid malignancy, namely ALK-positive anaplastic large cell lymphoma. Results from these studies have provided highly useful insights into the mechanisms by which a constitutively tyrosine kinase, such as NPM-ALK, promotes tumorigenesis. Several previous publications have comprehensively summarized the advances in this field. In this review, we provide readers with a brief update on specific areas of NPM-ALK pathobiology. In the first part, the NPM-ALK/signal transducer and activator of transcription 3 (STAT3) signaling axis is discussed, with an emphasis on the existence of multiple biochemical defects that have been shown to amplify the oncogenic effects of this signaling axis. Specifically, findings regarding JAK3, SHP1 and the stimulatory effects of several cytokines including interleukin (IL)-9, IL-21 and IL-22 are summarized. New concepts stemming from recent observations regarding the functional interactions among the NPM-ALK/STAT3 axis, ß catenin and glycogen synthase kinase 3ß will be postulated. Lastly, new mechanisms by which the NPM-ALK/STAT3 axis promotes tumorigenesis, such as its modulations of Twist1, hypoxia-induced factor 1α, CD274, will be described. In the second part, we summarize recent data generated by mass spectrometry studies of NPM-ALK, and use MSH2 and heat shock proteins as examples to illustrate the use of mass spectrometry data in stimulating new research in this field. In the third part, the evolving field of microRNA in the context of NPM-ALK biology is discussed.

Amot130 adapts atrophin-1 interacting protein 4 to inhibit yes-associated protein signaling and cell growth.

The adaptor protein Amot130 scaffolds components of the Hippo pathway to promote the inhibition of cell growth. This study describes how Amot130 through binding and activating the ubiquitin ligase AIP4/Itch achieves these effects. AIP4 is found to bind and ubiquitinate Amot130 at residue Lys-481. This both stabilizes Amot130 and promotes its residence at the plasma membrane. Furthermore, Amot130 is shown to scaffold a complex containing overexpressed AIP4 and the transcriptional co-activator Yes-associated protein (YAP). Consequently, Amot130 promotes the ubiquitination of YAP by AIP4 and prevents AIP4 from binding to large tumor suppressor 1. Amot130 is found to reduce YAP stability. Importantly, Amot130 inhibition of YAP dependent transcription is reversed by AIP4 silencing, whereas Amot130 and AIP4 expression interdependently suppress cell growth. Thus, Amot130 repurposes AIP4 from its previously described role in degrading large tumor suppressor 1 to the inhibition of YAP and cell growth.

Disheveled proteins promote cell growth and tumorigenicity in ALK-positive anaplastic large cell lymphoma.

Our previous oligonucleotide array studies revealed that ALK-positive anaplastic large cell lymphoma (ALK(+)ALCL) express high levels of the disheveled proteins (Dvls), a family of proteins that is integral to the Wnt signaling pathways. In this study, we assessed whether the Dvls are important in the pathogenesis of ALK(+)ALCL. By Western blotting, Dvl-2 and Dvl-3 were found to be highly expressed in ALK(+)ALCL cell lines and patient samples. The higher molecular weight forms, consistent with phosphorylated/active Dvl proteins, were observed in these lysates. siRNA knock-down of Dvls did not affect the Wnt canonical pathway, as assessed by the ß-catenin protein levels and nuclear localization. In contrast, the same treatment led to changes in the transcriptional activity of NFAT and the phosphorylation status of Src, both of which are known to be regulated by the Wnt non-canonical signaling pathways in other cell types. Coupled with these biochemical changes, there was a significant decrease in cell growth and soft agar colony formation. NPM-ALK, the oncogenic tyrosine kinase characteristic of ALK(+)ALCL, was found to bind to the Dvls and enhance their tyrosine phosphorylation. In conclusion, our data suggest that the Dvls contribute to the pathogenesis of ALK(+)ALCL via signaling in the Wnt non-canonical pathways. To our knowledge, this is the first report demonstrating a physical and functional interaction between the Dvls and an oncogenic tyrosine kinase.

NPM-ALK: The Prototypic Member of a Family of Oncogenic Fusion Tyrosine Kinases.

Anaplastic lymphoma kinase (ALK) was first identified in 1994 with the discovery that the gene encoding for this kinase was involved in the t(2;5)(p23;q35) chromosomal translocation observed in a subset of anaplastic large cell lymphoma (ALCL). The NPM-ALK fusion protein generated by this translocation is a constitutively active tyrosine kinase, and much research has focused on characterizing the signalling pathways and cellular activities this oncoprotein regulates in ALCL. We now know about the existence of nearly 20 distinct ALK translocation partners, and the fusion proteins resulting from these translocations play a critical role in the pathogenesis of a variety of cancers including subsets of large B-cell lymphomas, nonsmall cell lung carcinomas, and inflammatory myofibroblastic tumours. Moreover, the inhibition of ALK has been shown to be an effective treatment strategy in some of these malignancies. In this paper we will highlight malignancies where ALK translocations have been identified and discuss why ALK fusion proteins are constitutively active tyrosine kinases. Finally, using ALCL as an example, we will examine three key signalling pathways activated by NPM-ALK that contribute to proliferation and survival in ALCL.

Identification of two novel phenotypically distinct breast cancer cell subsets based on Sox2 transcription activity.

Sox2 (sex-determining region Y-box protein 2) is a transcription factor regulating pluripotency in embryonic stem cells. Sox2 is aberrantly expressed in breast and other cancers, though its biological significance remains widely unexplored. To understand the significance of this aberrancy, we assessed the transcription activity of Sox2 in two Sox2-expressing breast cancer cell lines, MCF7 and ZR751, using a lentiviral Sox2 GFP reporter vector. Surprisingly, Sox2 transcription activity, as measured by GFP expression encoded in a Sox2 reporter construct, was detectable only in a small subset of cells in both cell lines. Purification of GFP+ cells (cells with Sox2 activity) and GFP- cells (cells without Sox2 activity) was enriched for two phenotypically distinct cell populations in both MCF7 and ZR751 cell lines. Specifically, GFP+ cells formed significantly more colonies in methylcellulose and more mammospheres in vitro compared to GFP- cells. These phenotypic differences are directly linked to Sox2 as siRNA knockdown of Sox2 in GFP+ cells abolished these abilities. To provide a mechanistic explanation to our observations, we performed gel shift and chromatin immunoprecipitation studies; Sox2 was found to bind to its DNA binding consensus sequence and the promoters of Cyclin D1 and Nanog (two known Sox2 downstream targets) only in GFP+ cells. GFP+ cells also up-regulated CD49f, phospho-GSK3ß, and ß-catenin. In summary, we have identified two novel phenotypically distinct cell subsets in two breast cancer cell lines based on their differential Sox2 transcription activity. We demonstrate that Sox2 transcription activity, and not its protein expression alone, underlies the tumorigenicity and cancer stem cell-like phenotypes in breast cancers.

The heat shock protein-90 co-chaperone, Cyclophilin 40, promotes ALK-positive, anaplastic large cell lymphoma viability and its expression is regulated by the NPM-ALK oncoprotein.

BACKGROUND: Anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma (ALK+ ALCL) is a T cell lymphoma defined by the presence of chromosomal translocations involving the ALK tyrosine kinase gene. These translocations generate fusion proteins (e.g. NPM-ALK) with constitutive tyrosine kinase activity, which activate numerous signalling pathways important for ALK+ ALCL pathogenesis. The molecular chaperone heat shock protein-90 (Hsp90) plays a critical role in allowing NPM-ALK and other signalling proteins to function in this lymphoma. Co-chaperone proteins are important for helping Hsp90 fold proteins and for directing Hsp90 to specific clients; however the importance of co-chaperone proteins in ALK+ ALCL has not been investigated. Our preliminary findings suggested that expression of the immunophilin co-chaperone, Cyclophilin 40 (Cyp40), is up-regulated in ALK+ ALCL by JunB, a transcription factor activated by NPM-ALK signalling. In this study we examined the regulation of the immunophilin family of co-chaperones by NPM-ALK and JunB, and investigated whether the immunophilin co-chaperones promote the viability of ALK+ ALCL cell lines. METHODS: NPM-ALK and JunB were knocked-down in ALK+ ALCL cell lines with siRNA, and the effect on the expression of the three immunophilin co-chaperones: Cyp40, FK506-binding protein (FKBP) 51, and FKBP52 examined. Furthermore, the effect of knock-down of the immunophilin co-chaperones, either individually or in combination, on the viability of ALK+ ALCL cell lines and NPM-ALK levels and activity was also examined. RESULTS: We found that NPM-ALK promoted the transcription of Cyp40 and FKBP52, but only Cyp40 transcription was promoted by JunB. We also observed reduced viability of ALK+ ALCL cell lines treated with Cyp40 siRNA, but not with siRNAs directed against FKBP52 or FKBP51. Finally, we demonstrate that the decrease in the viability of ALK+ ALCL cell lines treated with Cyp40 siRNA does not appear to be due to a decrease in NPM-ALK levels or the ability of this oncoprotein to signal. CONCLUSIONS: This is the first study demonstrating that the expression of immunophilin family co-chaperones is promoted by an oncogenic tyrosine kinase. Moreover, this is the first report establishing an important role for Cyp40 in lymphoma.

Aberrant expression of the transcriptional factor Twist1 promotes invasiveness in ALK-positive anaplastic large cell lymphoma.

The transcriptional factor Twist1 has been shown to play a key role in regulating epithelial mesenchymal transition, invasiveness and migratory properties in solid tumors. We found that Twist1 is aberrantly expressed in ALK-positive anaplastic large cell lymphoma (ALK+ALCL), a type of T-cell lymphoid malignancy. Using RT-PCR and Western blots, Twist1 was detectable in all 3 ALK+ALCL cell lines examined but absent in normal T-cells. By immunohistochemistry, Twist1 was detectable in all 10 cases of ALK+ALCL examined; benign lymphoid tissues were consistently negative. Twist1 expression in ALK+ALCL cells can be attributed to the NPM-ALK/STAT3 signaling axis, the key oncogenic driving force in this tumor type. Twist1 is biologically important in ALK+ALCL cells, as Twist1 knockdown resulted in a significant decrease in their invasiveness in an in-vitro assay. Further investigation revealed that this increase in invasiveness is linked to the activation of AKT and down-regulation of p66Shc, two signaling proteins known to be involved in NPM-ALK-mediated oncogenesis. Lastly, knockdown of Twist1 sensitizes ALK+ALCL cells to the growth inhibitory effect of PF-2341066 (Crizotinib®), an ALK inhibitor being used in clinical trials. In conclusion, Twist1 expression, owing to the abnormal NPM-ALK/STAT3 signaling, contributes to its invasiveness and decreased sensitivity to PF-2341066 in ALK+ALCL.

NPM-ALK and the JunB transcription factor regulate the expression of cytotoxic molecules in ALK-positive, anaplastic large cell lymphoma.

Anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma (ALK+ ALCL) is an aggressive non-Hodgkin lymphoma of T/null immunophenotype that is most prevalent in children and young adults. The normal cellular counterpart of this malignancy is presumed to be the cytotoxic T lymphocyte (CTL), and this presumption is partly based on the observation that these tumour cells often express cytotoxic granules containing Granzyme B (GzB) and Perforin. Chromosomal translocations involving the gene encoding for the ALK tyrosine kinase are also characteristic of ALK+ ALCL, and the resulting fusion proteins (e.g. NPM-ALK) initiate signalling events important in ALK+ ALCL pathogenesis. These events include the elevated expression of JunB; an AP-1 family transcription factor that promotes ALK+ ALCL proliferation. In this report we demonstrate that JunB is a direct transcriptional activator of GzB and that GzB transcription is also promoted by NPM-ALK. We found that Perforin expression was not regulated by JunB, but was promoted by NPM-ALK in some cell lines and inhibited by it in others. In conclusion, our study makes the novel observation that signalling through NPM-ALK and JunB affect the expression of cytotoxic molecules in ALK+ ALCL. Moreover, these findings demonstrate the expression of GzB and Perforin in this lymphoma is not solely due its presumed CTL origin, but that oncogenic signalling is actively influencing the expression of these proteins.

Serine phosphorylation of NPM-ALK, which is dependent on the auto-activation of the kinase activation loop, contributes to its oncogenic potential.

It is well established that the tumorigenic potential of nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK), an oncogenic tyrosine kinase, is dependent on its tyrosine phosphorylation. Using tandem affinity purification-mass spectrometry, we found evidence of phosphorylation of three serine residues of NPM-ALK (Serine¹³5, Serine¹64 and Serine497) ectopically expressed in GP293 cells. Using a specific anti-phosphoserine antibody and immunoprecipitation, we confirmed the presence of serine phosphorylation of NPM-ALK in all three NPM-ALK-expressing cell lines examined. Similar to the tyrosine phosphorylation, phosphorylation of these serine residues was dependent on the activation status of the kinase activation loop of ALK. All of these three serine residues are biologically important as mutation of any one of these residues resulted in a significant reduction in the tumorigenicity of NPM-ALK (assessed by cell viability and clonogenic assay), which correlated with a substantial reduction in the phosphorylation of extracellular signal-regulated kinase 1/2, c-jun N-terminal kinase and signal transducer and activator of transcription 6. Serine phosphorylation of NPM-ALK appears to be regulated by multiple serine kinases since it was markedly reduced by pharmacologic inhibitors for glycogen synthase kinase-3, casein kinase I or mitogen-activated protein kinases. In summary, our study is the first to identify serine phosphorylation of NPM-ALK and to provide evidence that it enhances the tumorigenic potential of this oncogenic protein.

The tyrosine 343 residue of nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) is important for its interaction with SHP1, a cytoplasmic tyrosine phosphatase with tumor suppressor functions.

The cytoplasmic tyrosine phosphatase SHP1 has been shown to inhibit the oncogenic fusion protein nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK), and loss of SHP1 contributes to NPM-ALK-mediated tumorigenesis. In this study, we aimed to further understand how SHP1 interacts and regulates NPM-ALK. We employed an in vitro model in which GP293 cells were transfected with various combinations of NPM-ALK (or mutants) and SHP1 (or mutants) expression vectors. We found that SHP1 co-immunoprecipitated with NPM-ALK, but not the enzymatically inactive NPM-ALK(K210R) mutant, or the mutant in which all three functionally important tyrosine residues (namely, Tyr(338), Tyr(342), and Tyr(343)) in the kinase activation loop (KAL) of ALK were mutated. Interestingly, whereas mutation of Tyr(338) or Tyr(342) did not result in any substantial change in the NPM-ALK/SHP1 binding (assessed by co-immunoprecipitation), mutation of Tyr(343) abrogated this interaction. Furthermore, the NPM-ALK/SHP1 binding was readily detectable when each of the remaining 8 tyrosine residues known to be phosphorylated were mutated. Although the expression of SHP1 effectively reduced the level of tyrosine phosphorylation of NPM-ALK, it did not affect that of the NPM-ALK(Y343F) mutant. In soft agar clonogenic assay, SHP1 expression significantly reduced the tumorigenicity of NPM-ALK but not that of NPM-ALK(Y343F). In conclusion, we identified Tyr(343) of NPM-ALK as the crucial site for mediating the NPM-ALK/SHP1 interaction. Our results also support the notion that the tumor suppressor effects of SHP1 on NPM-ALK are dependent on its ability to bind to this oncogenic protein.

Alpha-sarcin catalytic activity is not required for cytotoxicity.

BACKGROUND: Alpha-sarcin is a protein toxin produced by Aspergillus giganteus. It belongs to a family of cytotoxic ribonucleases that inactivate the ribosome and inhibit protein synthesis. alpha-Sarcin cleaves a single phosphodiester bond within the RNA backbone of the large ribosomal subunit, which makes the ribosome unrecognizable to elongation factors and, in turn, blocks protein synthesis. Although it is widely held that the protein synthesis inhibition caused by the toxin leads to cell death, it has not been directly shown that catalytically inactive mutants of alpha-sarcin are non-toxic when expressed directly within the cytoplasm of cells. This is important since recent studies have cast doubt on whether protein synthesis inhibition is sufficient to initiate apoptosis. RESULTS: In this report, we assay alpha-sarcin cytotoxicity and ability to inhibit protein synthesis by direct cytoplasmic expression. We show that mutations in alpha-sarcin, which impair alpha-sarcin's ability to inhibit protein synthesis, do not affect its cytotoxicity. The mutants are unable to activate JNK, confirming that the sarcin-ricin loop remains intact and that the alpha-sarcin mutants are catalytically inactive. In addition, both mutant and wildtype variants of alpha-sarcin localize to the nucleus and cytoplasm, where they co-localize with ribosomal marker RPS6. CONCLUSION: We conclude that although protein synthesis inhibition likely contributes to cell death, it is not required. Thus, our results suggest that alpha-sarcin can promote cell death through a previously unappreciated mechanism that is independent of rRNA cleavage and JNK activation.

The K15 protein of Kaposi's sarcoma-associated herpesvirus recruits the endocytic regulator intersectin 2 through a selective SH3 domain interaction.

Kaposi's sarcoma-associated herpesvirus, also known as human herpesvirus 8, is closely associated with several cancers including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The rightmost end of the KSHV genome encodes a protein, K15, with multiple membrane-spanning segments and an intracellular carboxy-terminal tail that contains several conserved motifs with the potential to recruit interaction domains (i.e., SH2, SH3, TRAF) of host cell proteins. K15 has been implicated in downregulating B cell receptor (BCR) signaling through its conserved motifs and may thereby play a role in maintaining viral latency and/or preventing apoptosis of the infected B cells. However, K15's mode of action is largely unknown. We have used mass spectrometry, domain and peptide arrays, and surface plasmon resonance to identify binding partners for a conserved proline-rich sequence (PPLP) in the K15 cytoplasmic tail. We show that the PPLP motif selectively binds the SH3-C domain of an endocytic adaptor protein, Intersectin 2 (ITSN2). This interaction can be observed both in vitro and in cells, where K15 and ITSN2 colocalize to discrete compartments within the B cell. The ability of K15 to associate with ITSN2 suggests a new role for the K15 viral protein in intracellular protein trafficking.

Identification of a novel inhibitory actin-capping protein binding motif in CD2-associated protein.

CD2-associated protein (CD2AP) is a scaffold molecule that plays a critical role in the maintenance of the kidney filtration barrier. Little, however, is understood about its mechanism of function. We used mass spectrometry to identify CD2AP-interacting proteins. Many of the proteins that we identified suggest a role for CD2AP in endocytosis and actin regulation. To address the role of CD2AP in regulation of the actin cytoskeleton, we focused on characterizing the interaction of CD2AP with actin-capping protein CP. We identified a novel binding motif LXHXTXXRPK(X)6P present in CD2AP that is also found in its homolog Cin85 and other capping protein-associated proteins such as CARMIL and CKIP-1. CD2AP inhibits the function of capping protein in vitro. Therefore, our results support a role of CD2AP in the regulation of the actin cytoskeleton.