HIV: master of the host cell.

The human immunodeficiency virus has evolved various mechanisms to exploit its host cells, including the interruption and augmentation of signal transduction pathways. Recently, two DNA microarray studies have illustrated a remarkably broad-based perturbation in host transcriptional responses, which is in part mediated by the HIV-encoded Nef protein. HIV therefore seems to function as a 'master regulator' of cellular gene expression.

PKC-theta is required for TCR-induced NF-kappaB activation in mature but not immature T lymphocytes.

Productive interaction of a T lymphocyte with an antigen-presenting cell results in the clustering of the T-cell antigen receptor (TCR) and the recruitment of a large signalling complex to the site of cell-cell contact. Subsequent signal transduction resulting in cytokine gene expression requires the activation of one or more of the multiple isoenzymes of serine/threonine-specific protein kinase C (PKC). Among the several PKC isoenzymes expressed in T cells, PKC-theta is unique in being rapidly recruited to the site of TCR clustering. Here we show that PKC-theta is essential for TCR-mediated T-cell activation, but is dispensable during TCR-dependent thymocyte development. TCR-initiated NF-kappaB activation was absent from PKC-theta(-/-) mature T lymphocytes, but was intact in thymocytes. Activation of NF-kappaB by tumour-necrosis factor alpha and interleukin-1 was unaffected in the mutant mice. Although studies in T-cell lines had suggested that PKC-theta regulates activation of the JNK signalling pathway, induction of JNK was normal in T cells from mutant mice. These results indicate that PKC-theta functions in a unique pathway that links the TCR signalling complex to the activation of NF-kappaB in mature T lymphocytes.

Two distinct domains of the beta-subunit of glucosidase II interact with the catalytic alpha-subunit.

Recent purification and cDNA cloning of the endoplasmic reticulum processing enzyme glucosidase II have revealed that it is composed of two soluble proteins: a catalytic alpha-subunit and a beta-subunit of unknown function, both of which are highly conserved in mammals. Since the beta-subunit, which contains a C-terminal His-Asp-Glu-Leu (HDEL) motif, may function to link the catalytic subunit to the KDEL receptor as a retrieval mechanism, we sought to map the regions of the mouse beta-subunit protein responsible for mediating the association with the alpha-subunit. By screening a panel of recombinant beta-subunit glutathione S-transferase fusion proteins for the ability to precipitate glucosidase II activity, we have identified two non-overlapping interaction domains (ID1 and ID2) within the beta-subunit. ID1 encompasses 118 amino acids at the N-terminus of the mature polypeptide, spanning the cysteine-rich element in this region. ID2, located near the C-terminus, is contained within amino acids 273-400, a region occupied in part by a stretch of acidic residues. Variable usage of 7 alternatively spliced amino acids within ID2 was found not to influence the association of the two sub-units. We theorize that the catalytic subunit of glucosidase II binds synergistically to ID1 and ID2, explaining the high associative stability of the enzyme complex.

Use of chemokine receptors by poxviruses.

Chemokine receptors serve as portals of entry for certain intracellular pathogens, most notably human immunodeficiency virus (HIV). Myxoma virus is a member of the poxvirus family that induces a lethal systemic disease in rabbits, but no poxvirus receptor has ever been defined. Rodent fibroblasts (3T3) that cannot be infected with myxoma virus could be made fully permissive for myxoma virus infection by expression of any one of several human chemokine receptors, including CCR1, CCR5, and CXCR4. Conversely, infection of 3T3-CCR5 cells can be inhibited by RANTES, anti-CCR5 polyclonal antibody, or herbimycin A but not by monoclonal antibodies that block HIV-1 infection or by pertussis toxin. These findings suggest that poxviruses, like HIV, are able to use chemokine receptors to infect specific cell subtypes, notably migratory leukocytes, but that their mechanisms of receptor interactions are distinct.

Alternative splicing of transcripts encoding the alpha- and beta-subunits of mouse glucosidase II in T lymphocytes.

Glucosidase II is a processing enzyme of the endoplasmic reticulum that functions to hydrolyze two glucose residues in immature N -linked oligosaccharides attached to newly synthesized polypeptides. We previously reported the cDNA cloning of the alpha- and beta-subunits of mouse glucosidase II from T cells following copurification of these proteins with the highly glycosylated transmembrane protein-tyrosine phosphatase CD45. Subsequent examination of additional cDNA clones, coupled with partial genomic DNA sequencing, has revealed that both subunits are encoded by gene products that undergo alternative splicing in T lymphocytes. The catalytic alpha-subunit possesses two variably expressed segments, box Alpha1, consisting of 22 amino acids located proximal to the amino-terminus, and box Alpha2, composed of 9 amino acids situated between the amino-terminus and the putative catalytic site in the central region of the molecule. Box Beta1, a variably expressed 7 amino acid segment in the beta-subunit of glucosidase II, is located immediately downstream of an acidic stretch near the carboxyl-terminus. Screening of reverse transcribed RNA by polymerase chain reaction confirms the variable inclusion of each of these segments in transcripts obtained from a panel of T-lymphocyte cell lines. Thus, distinct isoforms of glucosidase II exist that may perform specialized functions.

Paxillin phosphorylation and association with Lck and Pyk2 in anti-CD3- or anti-CD45-stimulated T cells.

Antibodies to either CD3 or CD45 have been shown to induce dramatic changes in cell morphology, increased tyrosine phosphorylation of cellular proteins, and the association of a subset of these proteins with the tyrosine kinase Lck. The current study was initiated to determine the identity of the tyrosine-phosphorylated 70-80 kDa protein that becomes Lck-associated after stimulation with anti-CD45 or anti-CD3. We demonstrate that the cytoskeletal protein paxillin becomes tyrosine-phosphorylated when cells are plated on immobilized antibodies specific for CD45 or CD3. Only tyrosine-phosphorylated paxillin is associated with Lck, suggesting that the association is through the SH2 domain of Lck. Consistent with this we demonstrate that the SH2 domain of Lck binds tyrosine-phosphorylated paxillin. In contrast, the association of paxillin with the FAK-related kinase Pyk2 was found to be constitutive and not altered by the phosphorylation of either protein. Finally, we establish that the phosphorylation of paxillin is dependent on the expression of Lck. Taken together, these results demonstrate that paxillin is physically associated with kinases from two different families in T cells and suggest that paxillin may function as an adaptor protein linking cellular signals with cytoskeletal changes during T cell activation.

Identification of the CD45-associated 116-kDa and 80-kDa proteins as the alpha- and beta-subunits of alpha-glucosidase II.

CD45 is an abundant, highly glycosylated transmembrane protein-tyrosine phosphatase expressed on hematopoietic cells. Herein we demonstrate that two proteins of 116 kDa and 80 kDa copurify with CD45 from mouse T cells. Microsequence analysis of the 116-kDa protein revealed high similarity to an incomplete human open reading frame that has been suggested to correspond to the catalytic alpha-subunit of glucosidase II. We determined the nucleotide sequence of the mouse cDNA and observed that it encodes a protein product nearly identical to its human homologue and shares an active site consensus sequence with Family 31 glucosidases. Amino acid sequencing of the 80-kDa protein, followed by molecular cloning, revealed high homology to human and bovine cDNAs postulated to encode the beta-subunit of glucosidase II. Antisera developed to the mouse beta-subunit allowed us to demonstrate that the interaction between CD45 and glucosidase II can be reconstituted in vitro in an endoglycosidase H-sensitive manner. The strong interaction between glucosidase II and CD45 may provide a paradigm for investigating novel aspects of the biology of these proteins.

Immobilized antibodies to CD45 induce rapid morphologic changes and increased tyrosine phosphorylation of p56lck-associated proteins in T cells.

CD45 is a transmembrane protein tyrosine phosphatase required for signal transduction through the Ag receptor complexes of T and B lymphocytes. Herein, we demonstrate that immobilized mAbs to the external domain of CD45 induce rapid and dramatic morphologic changes in a variety of T cell lines, including CD8+ cytotoxic clones. CD45-induced morphologic changes can be inhibited by the cytoskeletal inhibitors cytochalasin D and E and by the protein tyrosine kinase inhibitor herbimycin A. Consistent with the requirement for tyrosine kinase activity, tyrosine phosphorylation of proteins at about 60 to 75 kDa and 115 to 130 kDa is increased upon engagement with immobilized anti-CD45 mAb with kinetics paralleling the observed changes in morphology. The phosphorylation of these proteins is inhibited by tyrosine kinase inhibitors at concentrations that also inhibit changes in morphology. The phosphoproteins induced when cells are added to immobilized anti-CD45 are co-immunoprecipitated with p56lck, suggesting that this tyrosine kinase might play a role in the phosphorylation of these proteins. Consistent with this, there is no increase in the phosphorylation of these proteins in p56lck-deficient CTLL-2 cells in response to immobilized anti-CD45 mAb. An important role for p56lck in the morphologic pathway is further supported by the observation that p56lck-deficient human J.CAM1.6 cells, in contrast to the parental Jurkat line, cannot be induced to undergo morphologic changes. Taken together, these results suggest a possible role for CD45 in coordinating a cytoskeletal remodeling cascade that may be important in cell activation.

CD45 protein-tyrosine phosphatase is specifically associated with a 116-kDa tyrosine-phosphorylated glycoprotein.

CD45 is a protein-tyrosine phosphatase expressed on all cells of hematopoietic origin. In an attempt to further characterize CD45 function, we set out to identify molecule(s) that specifically associate with CD45. A 116-kDa protein was detected in immunoprecipitates from CD45+ cells but not CD45- cells. The association between CD45 and this 116-kDa protein can be reconstituted by mixing lysates from CD45- cell lines with purified CD45. p116 appears to associate with CD45 through the external, transmembrane, or membrane-proximal region of CD45 since p116 is associated with a mutant form of CD45 possessing a truncated cytoplasmic domain. The association of p116 with CD45 is not isoform-specific as p116 associates equally well with various CD45 isoforms. We have determined that p116 is a tyrosine-phosphorylated glycoprotein and that it is associated with CD45 in all hematopoietic cells examined. Because of its broad distribution, it is possible that identification of p116 will provide additional insight into the function of CD45 in lymphoid as well as non-lymphoid hematopoietic cells.