Complementary methods for studies of protein interactions in living cells.
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Cell Physiological Phenomena↗
Biomedical subjects
Tom K Kerppola
Publications and source records attributed to Tom K Kerppola.
Visualization of molecular interactions by fluorescence complementation.
The visualization of protein complexes in living cells enables the examination of protein interactions in their normal environment and the determination of their subcellular localization. The bimolecular fluorescence complementation assay has been used to visualize interactions among multiple proteins in many cell types and organisms. Modified forms of this assay have been used to visualize the competition between alternative interaction partners and the covalent modification of proteins by ubiquitin-family peptides.
Microscopy, Fluorescence↗
Ubiquitin-mediated fluorescence complementation reveals that Jun ubiquitinated by Itch/AIP4 is localized to lysosomes.
Ubiquitin family peptide modifications regulate the functions and stabilities of many proteins. We have developed an approach for the visualization of ubiquitinated proteins in living cells designated ubiquitin-mediated fluorescence complementation (UbFC). This approach is based on complementation among fragments of fluorescent proteins when they are brought together by the covalent conjugation of ubiquitin fused to one fragment to a substrate protein fused to a complementary fragment. The UbFC strategy enables simultaneous visualization of proteins modified by different ubiquitin family peptides and comparison of their effects on protein localization. Visualization of ubiquitinated Jun revealed that it was localized predominantly to cytoplasmic structures. In contrast, Jun conjugated to small ubiquitin-related modifier 1 (SUMO1) was localized to subnuclear foci. Comparison of the distribution of ubiquitinated Jun with markers for various cytoplasmic compartments revealed that ubiquitinated Jun was localized to lysosomal vesicles. Fractionation of cell lysates confirmed that the majority of ubiquitinated Jun partitioned to the cytoplasmic fraction, and density gradient centrifugation analysis demonstrated that it cosedimented with lysosomal beta-hexosaminidase activity. Mutation of a recognition sequence for the E3 ligase Itch/AIP4 prevented Jun ubiquitination and stabilized it in cells. Inhibition of lysosomal protein degradation by bafilomycin or chloroquine stabilized Jun but had no effect on the stability of mutated Jun that was not ubiquitinated by Itch/AIP4. The visualization of ubiquitinated Jun in living cells has uncovered a lysosomal pathway for Jun degradation that involves ubiquitination by Itch/AIP4.
3T3 Cells↗
Visualization of Myc/Max/Mad family dimers and the competition for dimerization in living cells.
Myc and Mad family proteins play opposing roles in the control of cell growth and proliferation. We have visualized the subcellular locations of complexes formed by Myc/Max/Mad family proteins using bimolecular fluorescence complementation (BiFC) analysis. Max was recruited to different subnuclear locations by interactions with Myc versus Mad family members. Complexes formed by Max with Mxi1, Mad3, or Mad4 were enriched in nuclear foci, whereas complexes formed with Myc were more uniformly distributed in the nucleoplasm. Mad4 was localized to the cytoplasm when it was expressed separately, and Mad4 was recruited to the nucleus through dimerization with Max. The cytoplasmic localization of Mad4 was determined by a CRM1-dependent nuclear export signal located near the amino terminus. We compared the relative efficiencies of complex formation among Myc, Max, and Mad family proteins in living cells using multicolor BiFC analysis. Max formed heterodimers with the basic helix-loop-helix leucine zipper (bHLHZIP) domain of Myc (bMyc) more efficiently than it formed homodimers. Replacement of two amino acid residues in the leucine zipper of Max reversed the relative efficiencies of homo- and heterodimerization in cells. Surprisingly, Mad3 formed complexes with Max less efficiently than bMyc, whereas Mad4 formed complexes with Max more efficiently than bMyc. The distinct subcellular locations and the differences between the efficiencies of dimerization with Max indicate that Mad3 and Mad4 are likely to modulate transcription activation by Myc at least in part through distinct mechanisms.
Amino Acid Sequence↗
Synergistic transcription activation by Maf and Sox and their subnuclear localization are disrupted by a mutation in Maf that causes cataract.
Crystallin genes are selectively expressed during lens development. Maf and Sox family proteins synergistically enhanced gammaF-crystallin promoter activity in a lens cell line. Mutational analysis of the gammaF-crystallin promoter identified a composite regulatory element containing nonconsensus Maf and Sox recognition sequences. Mutations in these recognition sequences or changes in their spacing eliminated synergistic transcription activation. The transcriptional synergy was also affected by changes in the orientation of the Maf recognition sequence that had no detectable effect on binding affinity. The interaction between Maf and Sox proteins was visualized in living cells by bimolecular fluorescence complementation analysis. The N-terminal region of Maf mediated the interaction with Sox proteins in cells. Synergistic transcription activation required the N-terminal region of Maf as well as the ancillary DNA binding domain and the unique portion of the basic region that mediate specific recognition of the gammaF-crystallin promoter element. A mutation in the ancillary DNA binding domain of Maf (R288P) that has been shown to cause cataract eliminated the transcriptional activity of Maf but had no detectable effect on DNA binding in vitro. Whereas wild-type Maf was uniformly distributed in the nucleoplasm, R288P Maf was enriched in nuclear foci. Cajal bodies and gemini of coiled bodies were closely associated with the foci occupied by R288P Maf. Wild-type Maf formed complexes with Sox proteins in the nucleoplasm, whereas R288P Maf recruited Sox proteins as well as other interaction partners to the nuclear foci. The mislocalization of normal cellular proteins to these foci provides a potential explanation for the dominant disease phenotype of the R288P mutation in Maf.
Animals↗
Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis.
The specificity of biological regulatory mechanisms relies on selective interactions between different proteins in different cell types and in response to different extracellular signals. We describe a bimolecular fluorescence complementation (BiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. This approach is based on complementation between fragments of fluorescent proteins with different spectral characteristics. We have identified 12 bimolecular fluorescent complexes that correspond to 7 different spectral classes. Bimolecular complex formation between fragments of different fluorescent proteins did not differentially affect the dimerization efficiency of the bZIP domains of Fos and Jun or the subcellular sites of interactions between these domains. Multicolor BiFC enables visualization of interactions between different proteins in the same cell and comparison of the efficiencies of complex formation with alternative interaction partners.
Animals↗
The F-box protein Skp2 participates in c-Myc proteosomal degradation and acts as a cofactor for c-Myc-regulated transcription.
The transcription regulatory oncoprotein c-Myc controls genes involved in cell growth, apoptosis, and oncogenesis. c-Myc is turned over very quickly through the ubiquitin/proteasome pathway. The proteins involved in this process are still unknown. We have found that Skp2 interacts with c-Myc and participates in its ubiquitylation and degradation. The interaction between Skp2 and c-Myc occurs during the G1 to S phase transition of the cell cycle in normal lymphocytes. Surprisingly, Skp2 enhances c-Myc-induced S phase transition and activates c-Myc target genes in a Myc-dependent manner. Further, Myc-induced transcription was shown to be Skp2 dependent, suggesting interdependence between c-Myc and Skp2 in activation of transcription. Moreover, Myc-dependent association of Skp2, ubiquitylated proteins, and subunits of the proteasome to a c-Myc target promoter was demonstrated in vivo. The results suggest that Skp2 is a transcriptional cofactor for c-Myc and indicates a close relationship between transcription activation and transcription factor ubiquitination.
Animals↗
Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation.
Networks of protein interactions coordinate cellular functions. We describe a bimolecular fluorescence complementation (BiFC) assay for determination of the locations of protein interactions in living cells. This approach is based on complementation between two nonfluorescent fragments of the yellow fluorescent protein (YFP) when they are brought together by interactions between proteins fused to each fragment. BiFC analysis was used to investigate interactions among bZIP and Rel family transcription factors. Regions outside the bZIP domains determined the locations of bZIP protein interactions. The subcellular sites of protein interactions were regulated by signaling. Cross-family interactions between bZIP and Rel proteins affected their subcellular localization and modulated transcription activation. These results attest to the general applicability of the BiFC assay for studies of protein interactions.
3T3 Cells↗
Probing FinO-FinP RNA interactions by site-directed protein-RNA crosslinking and gelFRET.
The conjugative transfer of F-plasmids is repressed by a two-component system, which consists of the antisense RNA FinP and the protein FinO. FinO binds FinP, protecting it from endonucleolytic degradation and facilitating duplex formation between FinP and its complementary RNA. Here we present the results of site-specific protein-RNA cross-linking and gel-based fluorescence resonance energy transfer (gelFRET) experiments used to probe the structure of a complex of FinO bound to an RNA target consisting of a duplex with 5' and 3' single-stranded tails. The crosslinking experiments reveal that an extensive, largely positively charged surface on FinO contacts RNA. The gelFRET measurements indicate that the 5' single-stranded tail of the RNA is in closer contact with much of the protein than the distal, blunt end of the RNA duplex. These data suggest that significant conformational adjustments in the protein and/or the RNA accompany complex formation.
Bacterial Proteins↗