Subcellular micropatterning for visual immunoprecipitation reveals differences in cytosolic protein complexes downstream the EGFR

Analysis of protein-protein interactions in living cells by protein micropatterning is currently limited to the spatial arrangement of transmembrane proteins and their corresponding downstream molecules. Here we present a robust method for visual immunoprecipitation of cytosolic protein complexes by use of an artificial transmembrane bait construct in combination with micropatterned antibody arrays on cyclic olefin polymer (COP) substrates. The method was used to characterize Grb2-mediated signalling pathways downstream the epidermal growth factor receptor (EGFR). Ternary protein complexes (Shc1:Grb2:SOS1 and Grb2:Gab1:PI3K) were identified and we found that EGFR downstream signalling is based on constitutively bound (Grb2:SOS1 and Grb2:Gab1) as well as on agonist-dependent protein associations with transient interaction properties (Grb2:Shc1 and Grb2:PI3K). Spatiotemporal analysis further revealed significant differences in stability and exchange kinetics of protein interactions. Furthermore, we could show that this approach is well suited to study the efficacy and specificity of SH2 and SH3 protein domain inhibitors in a live cell context. Altogether, this method represents a significant enhancement of quantitative subcellular micropatterning approaches as an alternative to standard biochemical analyses.

CT7 (MAGE-C1) Promotes Survival and Interacts with STAT1 and PIASy in Multiple Myeloma Cells

Abstract 1807 The type I Melanoma Antigen GEnes MAGE-A3 and CT7 (MAGE-C1) are detected in more than 75% of primary multiple myeloma specimens, and their expression is correlated with proliferation and progression of disease. We previously showed that MAGE-A3 inhibits apoptosis in human myeloma cell lines (HMCL) and primary cells in part through ubiquitinylation of the prototypical tumor suppressor p53, which targets it for proteasomal degradation and inhibits its pro-apoptotic transcriptional program (Nardiello et al, Clin Cancer Res, 2011; 17:4309). However, silencing of MAGE-A3 in HMCL and primary cells that lacked functional p53, either through deletions or mutations, also resulted in apoptosis. Silencing of CT7 alone did not affect survival of HMCL that lacked p53, but it did increase their sensitivity to chemotherapy-induced apoptosis. Many type I MAGE proteins, including MAGE-A3, bind to the RING domain protein TRIM28/Kap1 through their highly conserved MAGE Homology Domain (MHD) to form E3 ubiquitin ligase complexes, but it was unknown if CT7 also associated with TRIM28. These results lead to the hypothesis that CT7 is a survival factor for myeloma cells that can act through p53-independent mechanisms. To investigate the biochemical activity of CT7 and identify non-p53 pathways regulated by type I MAGE in myeloma cells, we analyzed protein-protein interactions with CT7 by two methods. We immunoprecipitated (IP'ed) CT7 from lysates of HMCL followed by sequencing of co-IP'ed proteins by mass spectroscopy and we performed yeast two-hybrid screening with a bait construct containing the MHD of CT7. CT7 was reciprocally co-IP'ed from HMCL lysates with TRIM28, demonstrating that it, too, associated with this RING domain protein. These methods also revealed two novel interactions with CT7. CT7 reciprocally co-IP'ed with STAT1, a transcription factor that plays a critical role in receptor-mediated signaling for cytokines such as interferon α/β. CT7 also interacted with Protein Inhibitor of Activated STAT y (PIASy), a modified RING domain protein that negatively regulates STAT proteins through its Small Ubiquitin-like MOdifier (SUMO) ligase activity, which sequesters target proteins out of the nucleus and into the cytoplasm. Surprisingly, phosphorylated STAT1 (pSTAT1) was detected in lysates from unstimulated HMCL, indicating a tonic level of activation, but most or all of the pSTAT1 was in the cytoplasm, suggesting that its transcriptional activity was being blocked through exclusion from the nucleus. Phospho-STAT1 also appeared to preferentially associate with the CT7/TRIM28 complex. Phospho-STAT1 did not appear to be ubiquitinylated in these HMCL, but SUMO2/3 modification of pSTAT1 was detected. These results suggest that CT7 negatively regulates pSTAT1 activity in HMCL by sequestering the transcription factor out of the nucleus through SUMO modification. This may be a direct result of CT7-mediated SUMOylation in partnership with PIASy, or indirectly due to CT7/TRIM28-mediated ubiquitinylation events that result in activation of a SUMO ligase such as PIASy. These findings suggest the possibility that complexes containing type I MAGE proteins may also have SUMO ligase activity and identify STAT1 as a novel non-p53 biochemical pathway regulated by these genes. Disclosures: No relevant conflicts of interest to declare.

MAGI-1 interacts with Slo1 channel proteins and suppresses Slo1 expression on the cell surface

Large conductance Ca2+-activated K+ (BKCa) channels encoded by the Slo1 gene (also known as KCNMA1) are physiologically important in a wide range of cell types and form complexes with a number of other proteins that affect their function. We performed a yeast two-hybrid screen to identify proteins that interact with BKCa channels using a bait construct derived from domains in the extreme COOH-terminus of Slo1. A protein known as membrane-associated guanylate kinase with inverted orientation protein-1 (MAGI-1) was identified in this screen. MAGI-1 is a scaffolding protein that allows formation of complexes between certain transmembrane proteins, actin-binding proteins, and other regulatory proteins. MAGI-1 is expressed in a number of tissues, including podocytes and the brain. The interaction between MAGI-1 and BKCa channels was confirmed by coimmunoprecipitation and glutathione S-transferase pull-down assays in differentiated cells of a podocyte cell line and in human embryonic kidneys (HEK)293T cells transiently coexpressing MAGI-1a and three different COOH-terminal Slo1 variants. Coexpression of MAGI-1 with Slo1 channels in HEK-293T cells results in a significant reduction in the surface expression of Slo1, as assessed by cell-surface biotinylation assays, confocal microscopy, and whole cell recordings. Partial knockdown of endogenous MAGI-1 expression by small interfering RNA (siRNA) in differentiated podocytes increased the surface expression of endogenous Slo1 as assessed by electrophysiology and cell-surface biotinylation assays, whereas overexpression of MAGI-1a reduced steady-state voltage-evoked outward current through podocyte BKCa channels. These data suggest that MAGI-1 plays a role in regulation of surface expression of BKCa channels in the kidney and possibly in other tissues.