Identification of the novel K15 gene at the rightmost end of the Kaposi's sarcoma-associated herpesvirus genome

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a distinct open reading frame called K15 at a position equivalent to the gene encoding LMP2A of Epstein-Barr virus (EBV). K15 isolates from body cavity-based lymphoma (BCBL) cells exhibited a dramatic sequence variation and a complex splicing pattern. However, all K15 alleles are organized similarly with the potential SH2 and SH3 binding motifs in their cytoplasmic regions. Northern blot analysis showed that K15 was weakly expressed in latently infected BCBL-1 cells, and the level of its expression was significantly induced by tetradecanoyl phorbol acetate stimulation. K15 encoded 40- to 55-kDa proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was localized at the cytoplasm and plasma membrane. To demonstrate the signal-transducing activity of the K15 protein, we constructed a chimeric protein in which the cytoplasmic tail of the human CD8alpha polypeptide was replaced with that of KSHV K15. While the CD8-K15 chimera was not capable of eliciting cellular signal transduction upon stimulation with an anti-CD8 antibody, it significantly inhibited B-cell receptor signaling, as evidenced by a suppression of tyrosine phosphorylation and intracellular calcium mobilization. This inhibition required the putative SH2 or SH3 binding motif in the cytoplasmic region of K15. Biochemical study of CD8-K15 chimeras showed that the cytoplasmic region of K15 was constitutively tyrosine phosphorylated and that the tyrosine residue within the putative SH2 binding motif of K15 was a primary site of phosphorylation. These results demonstrate that KSHV K15 resembles LMP2A in genomic location, splicing pattern, and protein structure and by the presence of functional signal-transducing motifs in the cytoplasmic region. Thus, KSHV K15 is likely a distant evolutionary relative of EBV LMP2A.     

Identification of an Immunoreceptor Tyrosine-Based Activation Motif of K1 Transforming Protein of Kaposi’s Sarcoma-Associated Herpesvirus

Kaposi’s sarcoma-associated herpesvirus (KSHV) is consistently identified in Kaposi’s sarcoma and body cavity-based lymphoma. KSHV encodes a transforming protein called K1 which is structurally similar to lymphocyte receptors. We have found that a highly conserved region of the cytoplasmic domain of K1 resembles the sequence of immunoreceptor tyrosine-based activation motifs (ITAMs). To demonstrate the signal-transducing activity of K1, we constructed a chimeric protein in which the cytoplasmic tail of the human CD8α polypeptide was replaced with that of KSHV K1. Expression of the CD8-K1 chimera in B cells induced cellular tyrosine phosphorylation and intracellular calcium mobilization upon stimulation with an anti-CD8 antibody. Mutational analyses showed that the putative ITAM of K1 was required for its signal-transducing activity. Furthermore, tyrosine residues of the putative ITAM of K1 were phosphorylated upon stimulation, and this allowed subsequent binding of SH2-containing proteins. These results demonstrate that the KSHV transforming protein K1 contains a functional ITAM in its cytoplasmic domain and that it can transduce signals to induce cellular activation.     

Activation of mitogen-activated protein kinase and NF-kappaB pathways by a Kaposi's sarcoma-associated herpesvirus K15 membrane protein

The K15 gene of Kaposi's sarcoma-associated herpesvirus (also known as human herpesvirus 8) consists of eight alternatively spliced exons and has been predicted to encode membrane proteins with a variable number of transmembrane regions and a common C-terminal cytoplasmic domain with putative binding sites for SH2 and SH3 domains, as well as for tumor necrosis factor receptor-associated factors. These features are reminiscent of the latent membrane proteins LMP-1 and LMP2A of Epstein-Barr virus and, more distantly, of the STP, Tip, and Tio proteins of the related gamma(2)-herpesviruses herpesvirus saimiri and herpesvirus ateles. These viral membrane proteins can activate a number of intracellular signaling pathways. We have therefore examined the abilities of different K15-encoded proteins to initiate intracellular signaling. We found that a 45-kDa K15 protein derived from all eight K15 exons and containing 12 predicted transmembrane domains in addition to the cytoplasmic domain activated the Ras/mitogen-activated protein kinase (MAPK) and NF-kappaB pathways, as well as (more weakly) the c-Jun N-terminal kinase/SAPK pathway. Activation of the MAPK and NF-kappaB pathways required phosphorylation of tyrosine residue 481 within a putative SH2-binding site (YEEVL). This motif was phosphorylated by the tyrosine kinases Src, Lck, Yes, Hck, and Fyn. The region containing the YEEVL motif interacted with tumor necrosis factor receptor-associated factor 2 (TRAF-2), and a dominant negative TRAF-2 mutant inhibited the K15-mediated activation of the Ras/MAPK pathway, suggesting the involvement of TRAF-2 in the initiation of these signaling routes. In contrast, several smaller K15 protein isoforms activated these pathways only weakly. All of the K15 isoforms tested were, however, localized in lipid rafts, suggesting that incorporation into lipid rafts is not sufficient to initiate signaling. Additional regions of K15, located presumably in exons 2 to 5, may therefore contribute to the activation of these pathways. These findings illustrate that the 45-kDa K15 protein engages pathways similar to LMP1, LMP2A, STP, Tip, and Tio but combines functional features that are separated between LMP1 and LMP2A or STP and Tip.     

The M type K15 protein of Kaposi's sarcoma-associated herpesvirus regulates microRNA expression via its SH2-binding motif to induce cell migration and invasion

Kaposi's sarcoma (KS) associated herpesvirus (KSHV) is the etiological agent of KS. In vivo, KS is a tumor capable of spreading throughout the body, and pulmonary metastasis is observed clinically. In vitro, KSHV induces the invasiveness of endothelial cells. The KSHV open reading frame K15 is a KSHV-specific gene encoding a transmembrane protein. Two highly divergent forms of K15, the predominant (P) and minor (M) forms (K15P and K15M, respectively), have been identified in different KSHV strains. The two K15 alleles resemble the latent membrane protein 2A (LMP2A) gene of Epstein-Barr virus (EBV) in their genomic locations and protein topology. Also, both K15 proteins have motifs similar to those found in the EBV LMP1 protein. K15 therefore appears to be a hybrid of a distant evolutionary relative of EBV LMP1 and LMP2A. Since both LMP1 and LMP2A proteins are capable of inducing cell motility, we sought to determine whether K15 has similar abilities. In this study, we show that K15M is latently expressed in KSHV-positive PEL cells and knockdown of K15M in PEL cells reduces cell motility. K15M localizes to lysosomal membranes and induces cell migration, invasion, and NF-κB (but not AP-1) activity via its conserved SH2-binding motif. K15M also induces the expression of microRNAs miR-21 and miR-31 via this conserved motif, and knocking down both these microRNAs eliminates K15M-induced cell motility. Therefore, K15M may contribute to KSHV-mediated tumor metastasis and angiogenesis via regulation of miR-21 and miR-31, which we show here for the first time to be a specific regulator of cell migration. In light of these findings, the targeting of K15 or the downstream microRNAs regulated by it may represent novel therapies for treatment of KSHV-associated neoplasia.     

Intersectin 1 forms complexes with SGIP1 and Reps1 in clathrin-coated pits

Intersectin 1 (ITSN1) is an evolutionarily conserved adaptor protein involved in clathrin-mediated endocytosis, cellular signaling and cytoskeleton rearrangement. ITSN1 gene is located on human chromosome 21 in Down syndrome critical region. Several studies confirmed role of ITSN1 in Down syndrome phenotype. Here we report the identification of novel interconnections in the interaction network of this endocytic adaptor. We show that the membrane-deforming protein SGIP1 (Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1) and the signaling adaptor Reps1 (RalBP associated Eps15-homology domain protein) interact with ITSN1 in vivo. Both interactions are mediated by the SH3 domains of ITSN1 and proline-rich motifs of protein partners. Moreover complexes comprising SGIP1, Reps1 and ITSN1 have been identified. We also identified new interactions between SGIP1, Reps1 and the BAR (Bin/amphiphysin/Rvs) domain-containing protein amphiphysin 1. Immunofluorescent data have demonstrated colocalization of ITSN1 with the newly identified protein partners in clathrin-coated pits. These findings expand the role of ITSN1 as a scaffolding molecule bringing together components of endocytic complexes.     

Growth transformation of human T cells by herpesvirus saimiri requires multiple Tip-Lck interaction motifs

Lymphoma induction and T-cell transformation by herpesvirus saimiri strain C488 depends on two viral oncoproteins, StpC and Tip. The major interaction partner of Tip is the protein tyrosine kinase Lck, a key regulator of T-cell activation. The Lck binding domain (LBD) of Tip comprises two interaction motifs, a proline-rich SH3 domain-binding sequence (SH3B) and a region with homology to the C terminus of Src family kinase domains (CSKH). In addition, biophysical binding analyses with purified Lck-SH2 domain suggest the phosphorylated tyrosine residue 127 of Tip (pY127) as a potential third Lck interaction site. Here, we addressed the relevance of the individual binding motifs, SH3B, CSKH, and pY127, for Tip-Lck interaction and for human T-cell transformation. Both motifs within the LBD displayed Lck binding activities and cooperated to achieve a highly efficient interaction, while pY127, the major tyrosine phosphorylation site of Tip, did not enhance Lck binding in T cells. Herpesvirus saimiri strain C488 recombinants lacking one or both LBD motifs of Tip lost their transforming potential on human cord blood lymphocytes. Recombinant virus expressing Tip with a mutation at position Y127 was still able to transform human T lymphocytes but, in contrast to wild-type virus, was strictly dependent on exogenous interleukin-2. Thus, the strong Lck binding mediated by cooperation of both LBD motifs was essential for the transformation of human T cells by herpesvirus saimiri C488. The major tyrosine phosphorylation site Y127 of Tip was particularly required for transformation in the absence of exogenous interleukin-2, suggesting its involvement in cytokine signaling pathways.     

The LMP2A protein of Epstein–Barr virus regulates phosphorylation of ITSN1 and Shb adaptors by tyrosine kinases

Latent Membrane Protein 2A (LMP2A) is an Epstein–Barr virus-encoded protein that is important for the maintenance of latent infection. Its activity affects cellular differentiation, migration, proliferation and B cell survival. LMP2A resembles a constitutively activated B cell antigen receptor and exploits host kinases to activate a set of downstream signaling pathways. In the current study we demonstrate the interaction of LMP2A with intersectin 1 (ITSN1), a key endocytic adaptor protein. This interaction occurs via both the N- and C-tails of LMP2A and is mediated by the SH3 domains of ITSN1. Additionally, we identified the Shb adaptor and the Syk kinase as novel binding ligands of ITSN1. The Shb adaptor interacts simultaneously with the phosphorylated tyrosines of LMP2A and the SH3 domains of ITSN1 and mediates indirect interaction of ITSN1 to LMP2A. Syk kinase promotes phosphorylation of both ITSN1 and Shb adaptors in LMP2A-expressing cells. In contrast to ITSN1, Shb phosphorylation depends additionally on Lyn kinase activity.Considering that Shb and ITSN1 are implicated in various receptor tyrosine kinase signaling, our results indicate that LMP2A can affect a number of signaling pathways by regulating the phosphorylation of the ITSN1 and Shb adaptors.     

Activation of lymphocyte signaling by the R1 protein of rhesus monkey rhadinovirus

Rhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus that exhibits a considerable degree of similarity to the human Kaposi's sarcoma-associated herpesvirus (KSHV). The R1 protein of RRV is distantly related to the K1 protein of KSHV, and R1, like K1, can contribute to cell growth transformation. In this study we analyzed the ability of the cytoplasmic tail of R1 to function as a signal transducer. The cytoplasmic domain of the R1 protein contains several tyrosine residues whose phosphorylation is induced in cells expressing Syk kinase. Expression of a CD8 chimera protein containing the extracellular and transmembrane domains of CD8 fused to the cytoplasmic domain of R1 mobilized intracellular calcium and induced cellular tyrosine phosphorylation in B cells upon stimulation with anti-CD8 antibody. None of the CD8-R1 cytoplasmic deletion mutants tested were able to mobilize intracellular calcium or to induce tyrosine phosphorylation to a significant extent upon addition of anti-CD8 antibody. Expression of wild-type R1 protein activated nuclear factor of activated T lymphocytes (NFAT) eightfold in B cells in the absence of antibody stimulation; expression of the CD8-R1C chimera strongly induced NFAT activity (60-fold) but only upon the addition of anti-CD8 antibody. We conclude that the cytoplasmic domain of R1 is capable of transducing signals that elicit B-lymphocyte activation events. The signal-inducing properties of R1 appear to be similar to those of K1 but differ in that the required sequences are distributed over a much longer stretch of the cytoplasmic domain (>150 amino acids). In addition, the induction of calcium mobilization was considerably longer in duration and stronger with R1 than with K1.     

Alternative splicing affecting the SH3A domain controls the binding properties of intersectin 1 in neurons

Intersectin 1 (ITSN1) is a conserved adaptor protein implicated in endocytosis, regulation of actin cytoskeleton rearrangements and mitogenic signaling. Its expression is characterized by multiple alternative splicing. Here we show neuron-specific expression of ITSN1 isoforms containing exon 20, which encodes five amino acid residues in the first SH3 domain (SH3A). In vitro binding experiments demonstrated that inclusion of exon 20 changes the binding properties of the SH3A domain. Endocytic proteins dynamin 1 and synaptojanin 1 as well as GTPase-activating protein CdGAP bound the neuron-specific variant of the SH3A domain with higher affinity than ubiquitously expressed SH3A. In contrast, SOS1, a guanine nucleotide exchange factor for Ras, and the ubiquitin ligase Cbl mainly interact with the ubiquitously expressed isoform. These results demonstrate that alternative splicing leads to the formation of two pools of ITSN1 with potentially different properties in neurons, affecting ITSN1 function as adaptor protein.     

The N-terminal SH2 domains of Syk and ZAP-70 mediate phosphotyrosine-independent binding to integrin beta cytoplasmic domains

Syk and ZAP-70 form a subfamily of nonreceptor tyrosine kinases that contain tandem SH2 domains at their N termini. Engagement of these SH2 domains by tyrosine-phosphorylated immunoreceptor tyrosine-based activation motifs leads to kinase activation and downstream signaling. These kinases are also regulated by beta3 integrin-dependent cell adhesion via a phosphorylation-independent interaction with the beta3 integrin cytoplasmic domain. Here, we report that the interaction of integrins with Syk and ZAP-70 depends on the N-terminal SH2 domain and the interdomain A region of the kinase. The N-terminal SH2 domain alone is sufficient for weak binding, and this interaction is independent of tyrosine phosphorylation of the integrin tail. Indeed, phosphorylation of tyrosines within the two conserved NXXY motifs in the integrin beta3 cytoplasmic domain blocks Syk binding. The tandem SH2 domains of these kinases bind to multiple integrin beta cytoplasmic domains with varying affinities (beta3 (Kd = 24 nm) > beta2 (Kd = 38 nm) > beta1 (Kd = 71 nm)) as judged by both affinity chromatography and surface plasmon resonance. Thus, the binding of Syk and ZAP-70 to integrin beta cytoplasmic domains represents a novel phosphotyrosine-independent interaction mediated by their N-terminal SH2 domains.     

Structural analysis of the Kaposi's sarcoma-associated herpesvirus K1 protein

The K1 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) efficiently transduces extracellular signals to elicit cellular activation events through its cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM). In addition, the extracellular domain of K1 demonstrates regional homology with the immunoglobulin (Ig) family and contains conserved regions (C1 and C2) and variable regions (V1 and V2). To generate mouse monoclonal antibodies directed against the KSHV K1 protein, BALB/c mice were primed and given boosters with K1 protein purified from mammalian cells. Twenty-eight hybridomas were tested for reactivity with K1 protein by enzyme-linked immunosorbent assay, immunofluorescence, flow cytometry, immunohistochemistry, and immunoblotting. Deletion mutants of the K1 extracellular domain were used to map the epitope of each antibody. All antibodies were directed to the Ig, C1, and C2 regions of K1. Furthermore, antibody recognition of a short sequence (amino acids 92 to 125) of the C2 region overlapping with the Ig region of K1 efficiently induced intracellular free calcium mobilization; antibody recognition of the other regions of K1 did not. The efficient signal transduction of K1 induced by antibody stimulation required both the ITAM sequence of the cytoplasmic domain and the normal structure of the extracellular domain. Finally, immunological assays showed that K1 was expressed during the early lytic cycle of viral replication in primary effusion lymphoma cells. K1 was readily detected in multicentric Castleman's disease tissues, whereas it was not detected in Kaposi's sarcoma lesions, suggesting that K1 is preferentially expressed in lymphoid cells. Thus, these results indicate that the conserved regions, particularly the Ig and C2 regions, of the K1 extracellular domain are exposed on the outer surface and play an important role in K1 structure and signal transduction, whereas the variable regions of K1 appear to be away from the surface.     

Akt binds to and phosphorylates phospholipase C-gamma1 in response to epidermal growth factor

Both phospholipase (PL) C-gamma1 and Akt (protein kinase B; PKB) are signaling proteins that play significant roles in the intracellular signaling mechanism used by receptor tyrosine kinases, including epidermal growth factor (EGF) receptor (EGFR). EGFR activates PLC-gamma1 directly and activates Akt indirectly through phosphatidylinositol 3-kinase (PI3K). Many studies have shown that the PLC-gamma1 pathway and PI3K-Akt pathway interact with each other. However, it is not known whether PLC-gamma1 binds to Akt directly. In this communication, we identified a novel interaction between PLC-gamma1 and Akt. We demonstrated that the interaction is mediated by the binding of PLC-gamma1 Src homology (SH) 3 domain to Akt proline-rich motifs. We also provide a novel model to depict how the interaction between PLC-gamma1 SH3 domain and Akt proline-rich motifs is dependent on EGF stimulation. In this model, phosphorylation of PLC-gamma1 Y783 by EGF causes the conformational change of PLC-gamma1 to allow the interaction of its SH3 domain with Akt proline-rich motifs. Furthermore, we showed that the interaction between PLC-gamma1 and Akt resulted in the phosphorylation of PLC-gamma1 S1248 by Akt. Finally, we showed that the interaction between PLC-gamma1 and Akt enhanced EGF-stimulated cell motility.     

Identification of the R1 oncogene and its protein product from the rhadinovirus of rhesus monkeys

Rhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus that is most closely related to the human Kaposi's sarcoma-associated herpesvirus (KSHV). We have identified a distinct open reading frame at the left end of RRV and designated it R1. The position of the R1 gene is equivalent to that of the saimiri transforming protein (STP) of herpesvirus saimiri (HVS) and of K1 of KSHV, other members of the gamma-2 or rhadinovirus subgroup of herpesviruses. The R1 sequence revealed an open reading frame encoding a product of 423 amino acids that was predicted to contain an extracellular domain, a transmembrane domain, and a C-terminal cytoplasmic tail reflective of a type I membrane-bound protein. The predicted structural motifs of R1, including the presence of immunoreceptor tyrosine-based activation motifs, resembled those in K1 of KSHV but were distinct from those of STP. R1 sequences from four independent isolates from three different macaque species revealed 0.95 to 7.3% divergence over the 423 amino acids. Variation was located predominantly within the predicted extracellular domain. The R1 protein migrated at 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was extensively glycosylated. Tagged R1 protein was localized to the cytoplasmic and plasma membranes of transfected cells. Expression of the R1 gene in Rat-1 fibroblasts induced morphologic changes and focus formation, and injection of R1-expressing cells into nude mice induced the formation of multifocal tumors. A recombinant herpesvirus in which the STP oncogene of HVS was replaced by R1 immortalized T lymphocytes to interleukin-2-independent growth. These results indicate that R1 is an oncogene of RRV.     

Kaposi's sarcoma-associated herpesvirus K7 protein targets a ubiquitin-like/ubiquitin-associated domain-containing protein to promote protein degradation

Pathogens exploit host machinery to establish an environment that favors their propagation. Because of their pivotal roles in cellular physiology, protein degradation pathways are common targets for viral proteins. Protein-linking integrin-associated protein and cytoskeleton 1 (PLIC1), also called ubiquilin, contains an amino-terminal ubiquitin-like (UBL) domain and a carboxy-terminal ubiquitin-associated (UBA) domain. PLIC1 is proposed to function as a regulator of the ubiquitination complex and proteasome machinery. Kaposi's sarcoma-associated herpesvirus (KSHV) contains a small membrane protein, K7, that protects cells from apoptosis induced by various stimuli. We report here that cellular PLIC1 is a K7-interacting protein and that the central hydrophobic region of K7 and the carboxy-terminal UBA domain of PLIC1 are responsible for their interaction. Cellular PLIC1 formed a dimer and bound efficiently to polyubiquitinated proteins through its carboxy-terminal UBA domain, and this activity correlated with its ability to stabilize cellular I kappa B protein. In contrast, K7 interaction prevented PLIC1 from forming a dimer and binding to polyubiquitinated proteins, leading to the rapid degradation of I kappa B. Furthermore, K7 expression promoted efficient degradation of the p53 tumor suppressor, resulting in inhibition of p53-mediated apoptosis. These results indicate that KSHV K7 targets a regulator of the ubiquitin- and proteasome-mediated degradation machinery to deregulate cellular protein turnover, which potentially provides a favorable environment for viral reproduction.     

FBP WW domains and the Abl SH3 domain bind to a specific class of proline-rich ligands.

WW domains are conserved protein motifs of 38-40 amino acids found in a broad spectrum of proteins. They mediate protein-protein interactions by binding proline-rich modules in ligands. A 10 amino acid proline-rich portion of the morphogenic protein, formin, is bound in vitro by both the WW domain of the formin-binding protein 11 (FBP11) and the SH3 domain of Abl. To explore whether the FBP11 WW domain and Abl SH3 domain bind to similar ligands, we screened a mouse limb bud expression library for putative ligands of the FBP11 WW domain. In so doing, we identified eight ligands (WBP3 through WBP10), each of which contains a proline-rich region or regions. Peptide sequence comparisons of the ligands revealed a conserved motif of 10 amino acids that acts as a modular sequence binding the FBP11 WW domain, but not the WW domain of the putative signal transducing factor, hYAP65. Interestingly, the consensus ligand for the FBP11 WW domain contains residues that are also required for binding by the Abl SH3 domain. These findings support the notion that the FBP11 WW domain and the Abl SH3 domain can compete for the same proline-rich ligands and suggest that at least two subclasses of WW domains exist, namely those that bind a PPLP motif, and those that bind a PPXY motif.