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We have shown that the chemokine and HIV receptor CCR5 is palmitoylated on a cluster of cysteine residues located at the boundary between the seventh transmembrane region and the cytoplasmic tail. Single or combined substitutions of the three cysteines (Cys-321, Cys-323, and Cys-324) or incubation of wild-type CCR5-transfected cells with the palmitic acid analog 2-bromopalmitate prevented palmitoylation of the receptor. Moreover, failure of CCR5 to be palmitoylated resulted in both accumulation in intracellular stores and a profound decrease of membrane expression of the receptor. Upon metabolic labeling, kinetic experiments showed that the half-life of palmitoylation-deficient CCR5 is profoundly decreased. Bafilomycin A1, but not a specific proteasome inhibitor, prevented early degradation of palmitoylation-deficient CCR5 and promoted its accumulation in lysosomal compartments. Although membrane expression of the CCR5 mutant was diminished, the molecules reaching the membrane were still able to interact efficiently with the chemokine ligand MIP1 beta and remained able to function as HIV co-receptors. Thus we conclude that palmitoylation controls CCR5 expression through regulation of the life span of this receptor.

Signal-induced phosphorylation and ubiquitination of IkappaBalpha targets this inhibitor of NF-kappaB for proteasome-mediated degradation, thus permitting the release of active NF-kappaB. Upon cell stimulation, NF-kappaB activation results in neotranscription and neosynthesis of its own inhibitor, IkappaBalpha. As reported earlier, the neosynthesized inhibitor is then accumulated in the nucleus, where it rapidly binds to and terminates the function of nuclear NF-kappaB upon withdrawal of the stimulus. The present work was aimed at understanding how NF-kappaB activity is preserved while stimuli persist, despite intense, simultaneous IkappaBalpha neosynthesis, which would be expected to end NF-kappaB activity. We here show that incoming IkappaBalpha in the nucleus represents a target for resident nuclear proteasome complexes. Signal-induced, proteasome-dependent degradation of phosphorylated and ubiquitinated IkappaBalpha occurs in the nucleus, thus permitting the onset and persistence of NF-kappaB activity as long as stimulation is maintained. Our results suggest that intranuclear proteolysis of IkappaBalpha is necessarily required to avoid self-termination of NF-kappaB activity during cell activation.

Biological properties of chemokines are believed to be influenced by their association with glycosaminoglycans. Surface plasmon resonance kinetic analysis shows that the CXC chemokine stromal cell-derived factor-1alpha (SDF-1alpha), which binds the CXCR4 receptor, associates with heparin with an affinity constant of 38.4 nM (k(on) = 2.16 x 10(6) M(-1) s(-1) and k(off) = 0.083 x s(-1)). A modified SDF-1alpha (SDF-1 3/6) was generated by combined substitution of the basic cluster of residues Lys(24), His(25), and Lys(27) by Ser. SDF-1 3/6 conserves the global native structure and functional properties of SDF-1alpha, but it is unable to interact with sensor chip-immobilized heparin. The biological relevance of these in vitro findings was investigated. SDF-1alpha was unable to bind in a CXCR4-independent manner on epithelial cells that were treated with heparan sulfate (HS)-degrading enzymes or constitutively lack HS expression. The inability of SDF-1 3/6 to bind to cells underlines the importance of the identified basic cluster for the physiological interactions of SDF-1alpha with HS. Importantly, the amino-terminal domain of SDF-1alpha which is required for binding to, and activation of, CXCR4 remains exposed after binding to HS and is recognized by a neutralizing monoclonal antibody directed against the first residues of the chemokine. Overall, these findings indicate that the Lys(24), His(25), and Lys(27) cluster of residues forms, or is an essential part of, the HS-binding site which is distinct from that required for binding to, and signaling through, CXCR4.

Activation of NF-kappaB transcription factors requires phosphorylation and ubiquitin-proteasome-dependent degradation of IkappaB proteins. We provide evidence that a human F-box protein, h-betaTrCP, a component of Skp1-Cullin-F-box protein (SCF) complexes, a new class of E3 ubiquitin ligases, is essential for inducible degradation of IkappaBalpha. betaTrCP associates with Ser32-Ser36 phosphorylated, but not with unmodified IkappaBalpha or Ser32-Ser36 phosphorylation-deficient mutants. Expression of a F-box-deleted betaTrCP inhibits IkappaBalpha degradation, promotes accumulation of phosphorylated Ser32-Ser36 IkappaBalpha, and prevents NF-kappaB-dependent transcription. Our findings indicate that betaTrCP is the adaptor protein required for IkappaBalpha recognition by the SCFbetaTrCP E3 complex that ubiquitinates IkappaBalpha and makes it a substrate for the proteasome.

Recombinant interferon-gamma (IFN-gamma) induced HLA-DR gene expression in both U937 and THP-1 human monocytic cell lines, although the former was only very weakly inducible. Combination of recombinant tumor necrosis factor (TNF) and IFN-gamma resulted in a synergistic enhancement of DR mRNA and protein induction in both cell lines. TNF alone increased the constitutive expression of the DR gene in THP-1 cells. In the HLA class II-negative U937 cells, TNF used alone was not able to induce DR gene expression. Such a negative result was not due to a lack of TNF receptor expression in U937 cells, since TNF clearly induced HLA class I and TNF gene expression in this cell line. THP-1, but not U937, cells secreted TNF under the influence of IFN-gamma. Neutralization of TNF by a specific antibody decreased IFN-gamma-induced DR antigen expression in THP-1 cultures. These observations indicate that TNF is not able to directly induce DR gene expression, but rather amplifies ongoing expression of this gene, whether constitutive or induced by IFN-gamma. In the two cell lines tested, the level of DR inducibility under the influence of IFN-gamma used alone depended on a different inducibility of TNF secretion by IFN-gamma. Altogether, our observations indicate that TNF, whether exogenous or endogenously produced under the influence of IFN-gamma, amplifies DR gene expression in monocytes, a phenomenon that may provide to such antigen-presenting cells a selective sensitivity to the DR-inducing effects of IFN-gamma.

In unstimulated cells, the transcription factor NF-kappaB is held in the cytoplasm in an inactive state by the inhibitor protein IkappaBalpha. Stimulation of cells results in rapid phosphorylation and degradation of IkappaBalpha, thus releasing NF-kappaB, which translocates to the nucleus and activates transcription of responsive genes. Here we demonstrate that in cells where proteasomal degradation is inhibited, signal induction by tumor necrosis factor alpha results in the rapid accumulation of higher molecular weight forms of IkappaBalpha that dissociate from NF-kappaB and are consistent with ubiquitin conjugation. Removal of the high molecular weight forms of IkappaBalpha by a recombinant ubiquitin carboxyl-terminal hydrolase and reactivity of the immunopurified material with a monoclonal antibody specific for ubiquitin indicated that IkappaBalpha was conjugated to multiple copies of ubiquitin. Western blot analysis of immunopurified IkappaBalpha from cells expressing epitope-tagged versions of IkappaBalpha and ubiquitin revealed the presence of multiple copies of covalently bound tagged ubiquitin. An S32A/S36A mutant of IkappaBalpha that is neither phosphorylated nor degraded in response to signal induction fails to undergo inducible ubiquitination in vivo. Thus signal-induced activation of NF-kappaB involves phosphorylation-dependent ubiquitination of IkappaBalpha, which targets the protein for rapid degradation by the proteasome and releases NF-kappaB for translocation to the nucleus.

In rat lymph node lymphocytes stimulated for 24 h by concanavalin A in the presence of 10−5M 2′-deoxycoformycin (a potent inhibitor of adenosine deaminase) and 10−5M 2′-deoxyadenosine the adenylic nucleotide pool was reduced by 55.5% without modification of either the adenylic energy charge or the ability of the cells to liberate interleukin 2. In the same conditions, the ability of rat spleen cells to bind exogenous interleukin 2 activity was not modified. The proliferative response to concanavalin A stimulation was completely inhibited after a 86-h culture period under adenosine deaminase deficiency conditions. It could not be restored by elimination of 2′-deoxyadenosine after a 20-h pretreatment, when adenylic nucleotide pool depletion was 72.4% whereas the interleukin 2 liberation ability was not suppressed. These results suggest that among the early consequences of adenosine deaminase deficiency conditions, which occur before S phase of the cell cycle, the depletion of adenylic nucleotide pool, rather than the impairment of interleukin 2 liberation and absorption capacities, may account for the inability of the lymphocytes to respond to mitogenic stimulation.