33,34 We found that T-cell activation caused a 2·5-fold induction of SKP2 mRNA and a 6-fold induction of CKS1B, and the same occurred in cells exposed to nIL-2, BMS-345541

or PS-1145. Therefore, we conclude that, at the transcriptional level, SKP2 and CKS1B are not influenced by the functional status of IKK or IL-2 signalling. However, at the protein level, SKP2 and CKS1B expression was unaffected by nIL-2, but suppressed by BMS-345541 and PS-1145. Thus, we further conclude selleck chemicals that, in stimulated human naïve CD4+ T cells, IKK activation is crucial for the stability of the F-box protein SKP2 and its co-factor CKS1B. As phosphorylation of SKP2 on serine 64/72 is required for its stabilization Fluorouracil order and protection from anaphase-promoting complex (APC)Cdh1-mediated degradation,43,44 we propose that IKK activation assists, or is required for, this stabilizing mechanism in human T cells. Inhibition by BMS-345541 or PS-1145 appears to be specific, because expression of β-actin, β-tubulin, lamin-B1, GAPDH and proteasome subunit α5 was similar in costimulated T cells with and without pretreatment, which excludes a general block in protein expression by either drug. This was supported by the comparable levels of induction seen for the NFAT-regulated EGR-2 transcription factor.

While PS-1145 is essentially an IKKβ inhibitor with a 50% inhibitory concentration (IC50) of 0·15 μm, BMS-345541 can inhibit IKKβ and IKKα, although with different IC50s: 0·3 μm for IKKβ and 4 μm for IKKα.45 Therefore, the observations of the present study appear to result mainly from the inhibition of IKKβ, although the possibility of a contribution from IKKα inhibition cannot be formally excluded. BMS-345541 and PS-1145 are structurally unrelated, and share the unique, non-specific target, ERK-8 protein kinase.46 As this

is virtually absent in circulating leucocytes47 our results are presumably not caused by the inhibition of kinases other than IKK. Both the pharmacological inhibition of IKK48 and the genetic repression of NF-κB proteins through the expression of a dominant negative form of I-κBα49 are associated with markedly impaired proliferative ALOX15 responses of T cells, although the mechanisms by which this occurs are unclear. By demonstrating the ability of IKK-mediated signals to regulate transcription of cyclin D3, CDK2 and cyclin E, and protein stability of SKP2 and its co-factor CKS1B through IL-2-independent mechanisms, this study provides new information about the function of IKK in T-cell proliferation. However, with the exception of cyclin D3, no NF-κB binding sites have been reported in the promoters of the CDK2 or cyclin E genes. Therefore, no obvious explanation exists for the molecular mechanisms that link the pharmacological inhibition of IKK with the inhibition of CDK2 and cyclin E up-regulation in human T cells.