Post-translational modification by the conjugation of small ubiquitin-like modifiers is an essential mechanism to affect protein function. Currently, only a limited number of substrates are known for most of these modifiers, thus limiting our knowledge of their role and relevance for cellular physiology. Here, we report the development of a universal strategy for proteomic studies of ubiquitin-like modifiers. This strategy involves the development of stable transfected cell lines expressing a double-tagged modifier under the control of a tightly negatively regulated promoter, the induction of the expression and conjugation of the tagged modifier to cellular proteins, the tandem affinity purification of the pool of proteins covalently modified by the tagged modifier, and the identification of the modified proteins by LC and MS. By applying this methodology to the proteomic analysis of SUMO-1 and SUMO-3, we determined that SUMO-1 and SUMO-3 are stable proteins exhibiting half-lives of over 20 h, demonstrated that sumoylation with both SUMO-1 and SUMO-3 is greatly stimulated by MG-132 and heat shock treatment, demonstrated the preferential usage of either SUMO-1 or SUMO-3 for some known SUMO substrates, and identified 122 putative SUMO substrates of which only 27 appeared to be modified by both SUMO-1 and SUMO-3. This limited overlapping in the subset of proteins modified by SUMO-1 and SUMO-3 supports that the SUMO paralogues are likely to be functionally distinct. Three of the novel putative SUMO substrates identified, namely the polypyrimidine tract-binding protein-associated splicing factor PSF, the structural microtubular component ␣-tubulin, and the GTP-binding nuclear protein Ran, were confirmed as authentic SUMO substrates. The application of this universal strategy to the identification of the pool of cellular substrates modified by other ubiquitin-like modifiers will dramatically increase our knowledge of the biological role of the different ubiquitin-like conjugations systems in the cell. Molecular & Cellular Proteomics 4:56 -72, 2005.

In vitro models to study the process of keratinocyte differentiation have been hindered by the stringent culture requirements and limitations imposed by the inherent properties of the cells. Primary keratinocytes only have a finite life span, while transformed cell lines exhibit many phenotypic features not found in normal cells. The spontaneously immortalized HaCaT cell line has been a widely employed keratinocyte model due to its ease of propagation and near normal phenotype, but protocols for differentiation and gene delivery into HaCaT cells vary widely in the literature. Here we report culture conditions for maintaining HaCaT cells in a basal-like state, for efficient differentiation of these cells, and for delivery of transgenes by transfection or adenoviral infection. This technological report will provide guidance to a large audience of scientists interested in investigating mechanisms of differentiation and skin morphogenesis.

Regulation of BCR signalling strength is crucial for B-cell development and function. Bright is a B-cell-restricted factor that complexes with Bruton's tyrosine kinase (Btk) and its substrate, transcription initiation factor-I (TFII-I), to activate immunoglobulin heavy chain gene transcription in the nucleus. Here we show that a palmitoylated pool of Bright is diverted to lipid rafts of resting B cells where it associates with signalosome components. After BCR ligation, Bright transiently interacts with sumoylation enzymes, blocks calcium flux and phosphorylation of Btk and TFII-I and is then discharged from lipid rafts as a Sumo-I-modified form. The resulting lipid raft concentration of Bright contributes to the signalling threshold of B cells, as their sensitivity to BCR stimulation decreases as the levels of Bright increase. Bright regulates signalling independent of its role in IgH transcription, as shown by specific dominant-negative titration of rafts-specific forms. This study identifies a BCR tuning mechanism in lipid rafts that is regulated by differential post-translational modification of a transcription factor with implications for B-cell tolerance and autoimmunity.

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DNA binding regions I, II, and III at the origin of replication have different arrangements of A protein (T antigen) recognition pentanucleotides. The A protein also protects each region from DNase in distinctly different patterns. Footprint and fragment assays led to the following conclusions: (i) in some cases a single recognition pentanucleotide is sufficient to direct the binding and accurate alignment of A protein on DNA; (ii) the A protein binds within isolated region I or II in a sequential process leading to multiple overlapping areas of DNase protection within each region; and (iii) the 23-base pair span of recognition sequences in region II allows binding and protection of a longer length of DNA than the 23-base pair span in region I. We propose a model of protein binding that addresses the problem of variations in the arrangement of pentanucleotides in regions I and II and explains the observed DNase protection patterns. The central feature of the model requires each protomer of A protein to bind to a pentanucleotide in a unique direction. The resulting orientation of protein would protect more DNA at the 5' end of the 5'-GAGGC-3' recognition sequence than at the 3' end. The arrangement of multiple protomers at the origin of simian virus 40 replication is discussed. of PIPES binding buffer with 0.1 M MgCl2 and 0.05 M CaCl2 was added for 5 min at 0°C in 0.005 M NaCl.