Expanding the substantial interactome of NEMO using protein microarrays

Abstract

Signal transduction by the NF-kappaB pathway is a key regulator of a host of cellular responses to extracellular and intracellular messages. The NEMO adaptor protein lies at the top of this pathway and serves as a molecular conduit, connecting signals transmitted from upstream sensors to the downstream NF-kappaB transcription factor and subsequent gene activation. The position of NEMO within this pathway makes it an attractive target from which to search for new proteins that link NF-kappaB signaling to additional pathways and upstream effectors. In this work, we have used protein microarrays to identify novel NEMO interactors. A total of 112 protein interactors were identified, with the most statistically significant hit being the canonical NEMO interactor IKKbeta, with IKKalpha also being identified. Of the novel interactors, more than 30% were kinases, while at least 25% were involved in signal transduction. Binding of NEMO to several interactors, including CALB1, CDK2, SAG, SENP2 and SYT1, was confirmed using GST pulldown assays and coimmunoprecipitation, validating the initial screening approach. Overexpression of CALB1, CDK2 and SAG was found to stimulate transcriptional activation by NF-kappaB, while SYT1 overexpression repressed TNFalpha-dependent NF-kappaB transcriptional activation in human embryonic kidney cells. Corresponding with this finding, RNA silencing of CDK2, SAG and SENP2 reduced NF-kappaB transcriptional activation, supporting a positive role for these proteins in the NF-kappaB pathway. The identification of a host of new NEMO interactors opens up new research opportunities to improve understanding of this essential cell signaling pathway.

Figure 1. Probing of the human protein microarray with biotinylated recombinant NEMO.

(A) Domain structure of the human NEMO protein, showing the two coiled coil domains (CC1 and CC2), the NEMO ubiquitin binding domain (NUB), leucine zipper (LZ) and zinc finger (ZF). (B) Immunoblot detection of biotinylated NEMO following purification of GST-NEMO, cleavage of the GST tag and biotinylation. Biotinylated NEMO was detected with streptavidin-alkaline phosphatase conjugate. (C) Example hits obtained from the array, compared to the same spot positions on negative control array. (D) Frequency histogram for the NEMO-probed protein microarray showing the range of Z-scores obtained. Protein interactors with a Z-score greater than three (Z>3; P<0.002) were deemed significant. Scores obtained for the canonical NEMO interactors IKKalpha (Z = 6.52) and IKKbeta (Z = 8.41) are shown for reference. Scores were calculated using Invitrogen Protoarray Prospector version 5.1 software. See Table 1 for gene descriptions.

Figure 2. Cytoplasmic and nuclear signaling kinases dominate the NEMO interactome.

Gene ontologies were determined for each of the NEMO interactors and the results for each of the three standard ontological categories plotted as percentages. Genes belonging to more than one category were assigned to the category for which the gene has been best characterized.

Figure 3. Putative interactors bind to NEMO in GST pulldown, coimmunoprecipitation and mammalian two-hybrid assays.

(A) Immunoblot analysis of GST and GST-NEMO proteins used as control and bait for the pulldown assay. Proteins were detected using anti-GST/HRP conjugate following SDS-PAGE and membrane transfer. (B) Results of GST pulldown assays showing binding of NEMO to putative interactors identified by protein array screening. Each of the interactors and IKKbeta, a known NEMO binder, were overexpressed in transiently transfected HEK-293T cells and the resulting lysates applied to immobilized GST or GST-NEMO. Following incubation and washing, the samples were resolved by SDS-PAGE and the proteins detected using appropriate antibodies. Input lanes were loaded with 5–10% of HEK-293T lysates to confirm protein expression. The size of relevant protein markers is shown beside the blot image. (C–H) Coimmunoprecipitation assays between NEMO and putative binders in HEK-293T cells. Plasmids encoding Xpress-tagged NEMO or the empty parent vector and tagged putative binders were used to transfect HEK-293T cells and the resulting cell lysates used for coimmunoprecipitation assays. For each putative binder, immunoblots are shown for detection of the binder using a tag- or protein-specific antibody, and for detection of Xpress-tagged NEMO. For IKKbeta and each of the five putative interactors, substantial coimmunoprecipitation occurred only in the presence immunoprecipitated NEMO. Input lanes contained 5–10% of the precleared input volume used prior to addition of anti-Xpress antibody. Binding and washing steps were performed in the presence of 0.5% NP-40 for all proteins except SAG, where 0.1% NP-40 was used. (I) NEMO interacts with CALB1, CDK2, SAG, SENP2 and SYT1 in a mammalian two-hybrid system. Empty two-hybrid vectors were cotransfected as a negative control. The MyoD/Id and NEMO/IkappaBalpha protein pairs were used as positive controls, while putative interaction partners cotransfected with empty complementing vector were used as negative controls. For each pair tested, a significant increase (n = 6; two-tailed T test; P≤0.05) in luciferase activity was obtained in partner/NEMO experiments compared to partner/vector experiments (indicated by asterisks).

Figure 4. NEMO interactors influence the transcriptional activation activity NF-kappaB.

(A) Each of the five NEMO interactors was overexpressed in untreated HEK-293T cells and their effect on NF-kappaB transcriptional activation measured by a reporter assay. CALB1, CDK2 and SAG significantly increased reporter activity, indicated by asterisks (n = 4; two-tailed T test; P≤0.05), while other genes had no effect compared to the control vector transfection. Reporter activity is given in relative light units (RLU). (B) CALB1 and CDK2 overexpressed increases NF-kappaB activity in TNFalpha treated cells, while SYT1 overexpression significantly represses activity (n = 4; two-tailed T test; P≤0.05). (C) Confirmation of protein expression following transfection of HEK-293T cells by immunoblot detection of native or epitope-tagged NEMO interactors. Little or no protein expression was detected in the control vector transfected cells. (D) Knockdown of CDK2, SAG and SENP2 in HEK-293T cells mediated by siRNA transfection. RNA levels at the time of NF-kappaB assays were measured by RT-qPCR and are displayed as percentage mRNA remaining after knockdown compared to the amounts present in control siRNA-treated cells. Significant knockdown was seen for both of the genes, as indicated by the asterisks (n = 3; two-tailed T test; P≤0.05). (E) mRNA knockdown of CDK2, SAG and SENP2 reduces NF-kappaB transcriptional activation in TNFalpha stimulated HEK-293T cells, but does not impact upon basal NF-kappaB activity in untreated cells (n = 4; two-tailed T test; P≤0.05).