Conserved beta-hairpin recognition by the GYF domains of Smy2 and GIGYF2 in mRNA surveillance and vesicular transport complexes.

The yeast suppressor of myosin 2 protein (Smy2) interacts with mRNA-processing proteins through recognition of proline-rich sequences (PRS). Here, we describe the crystal structure of the GYF domain of Smy2 in association with a PRS from the yeast branch point binding protein (BBP/ScSF1). Complex formation requires that the beta-hairpin of the central PPGL motif of the ligand is accommodated by an extended hydrophobic cleft in the domain-a specificity feature that is maintained in the human protein GIGYF2. SILAC/MS experiments in combination with PRS site inhibition show that Smy2 associates with the Ccr4-NOT deadenylase complex, whereas GIGYF2 interacts not only with mRNA surveillance factors, but also with vesicular transport proteins and Atrophin-1. GIGYF2 is shown to associate with COPII-vesicle proteins and localize to the ER and Golgi in resting cells, whereas environmental challenge drives GIGYF2 into stress granules. The current study highlights the structural basis for PRS recognition by Smy2-type GYF domains, and implicates Smy2 and GIGYF2 in both mRNA processing and the secretory pathway.

A novel S3S-TAP-tag for the isolation of T-cell interaction partners of adhesion and degranulation promoting adaptor protein.

The identification of modular units of cellular function is a major goal for proteomic research. Protein complexes represent important building blocks defining functionality and deciphering their composition remains a major challenge. Here, we have designed a new tandem affinity purification (TAP) tag (termed S3S-tag) for the isolation of protein complexes. Specifically, the immune cell protein ADAP that regulates integrin adhesion was fused either C- or N-terminally to the S3S-tag. After retroviral transduction of a vector containing S3S-tagged ADAP and internal ribosomal entry site encoded enhanced green fluorescent protein (eGFP), Jurkat T cells were sorted according to eGFP expression and further selected for expression of TAP-tagged protein close to endogenous levels. The combination of a cleavable S-tag and a Strep-tag II allowed for the isolation of ADAP and associated proteins. Subsequently, stable isotope labeling with amino acids in cell culture-based mass spectrometric analysis was performed to identify potentially specific interaction partners. Co-purification of the known interaction partner Src kinase-associated phosphoprotein of 55 kDa indicates the validity of our approach, while the identification of the ENA/VASP family member EVL, the guanine nucleotide exchange factor GEF-H1 and the adaptor protein DOCK2 corroborates a link between ADAP-mediated integrin regulation and the cytoskeleton.

Proline-rich sequence recognition: I. Marking GYF and WW domain assembly sites in early spliceosomal complexes.

Proline-rich sequences (PRS) and their recognition domains have emerged as transposable protein interaction modules during eukaryotic evolution. They are especially abundant in proteins associated with pre-mRNA splicing and likely assist in the formation of the spliceosome by binding to GYF and WW domains. Here we profile PRS-mediated interactions of the CD2BP2/52K GYF domain by a site-specific peptide inhibitor and stable isotope labeling/mass spectrometry analysis. Several PRS hubs with multiple proline-rich motifs exist that can recruit GYF and/or WW domains. Saturating the PRS sites by an isolated GYF domain inhibited splicing at the level of A complex formation. The interactions mediated by PRS are therefore important to the early phases of spliceosomal assembly.

Proline-rich sequence recognition: II. Proteomics analysis of Tsg101 ubiquitin-E2-like variant (UEV) interactions.

The tumor maintenance protein Tsg101 has recently gained much attention because of its involvement in endosomal sorting, virus release, cytokinesis, and cancerogenesis. The ubiquitin-E2-like variant (UEV) domain of the protein interacts with proline-rich sequences of target proteins that contain P(S/T)AP amino acid motifs and weakly binds to the ubiquitin moiety of proteins committed to sorting or degradation. Here we performed peptide spot analysis and phage display to refine the peptide binding specificity of the Tsg101 UEV domain. A mass spectrometric proteomics approach that combines domain-based pulldown experiments, binding site inactivation, and stable isotope labeling by amino acids in cell culture (SILAC) was then used to delineate the relative importance of the peptide and ubiquitin binding sites. Clearly "PTAP" interactions dominate target recognition, and we identified several novel binders as for example the poly(A)-binding protein 1 (PABP1), Sec24b, NFkappaB2, and eIF4b. For PABP1 and eIF4b the interactions were confirmed in the context of the corresponding full-length proteins in cellular lysates. Therefore, our results strongly suggest additional roles of Tsg101 in cellular regulation of mRNA translation. Regulation of Tsg101 itself by the ubiquitin ligase TAL (Tsg101-associated ligase) is most likely conferred by a single PSAP binding motif that enables the interaction with Tsg101 UEV. Together with the results from the accompanying article (Kofler, M., Schuemann, M., Merz, C., Kosslick, D., Schlundt, A., Tannert, A., Schaefer, M., Lührmann, R., Krause, E., and Freund, C. (2009) Proline-rich sequence recognition: I. Marking GYF and WW domain assembly sites in early spliceosomal complexes. Mol. Cell. Proteomics 8, 2461-2473) on GYF and WW domain pathways our work defines major proline-rich sequence-mediated interaction networks that contribute to the modular assembly of physiologically relevant protein complexes.

In-Gel 18O labeling for improved identification of proteins from 2-DE Gel spots in comparative proteomic experiments.

The reliability of 2-DE gel-based comparative proteomics is severely impaired by the potential presence of overlapping proteins. We describe a methodological procedure which may solve this problem. Corresponding protein spots from two experimental groups are digested in the presence of 16O and 18O, respectively. Samples are pooled and proteins identified by MS. The 18O/16O-ratios of the different proteins found in the same spot distinguish proteins with altered from those whose intensity is unchanged.

PRMT1-mediated arginine methylation of PIAS1 regulates STAT1 signaling.

To elucidate the function of the transcriptional coregulator PRMT1 (protein arginine methyltranferase 1) in interferon (IFN) signaling, we investigated the expression of STAT1 (signal transducer and activator of transcription) target genes in PRMT1-depleted cells. We show here that PRMT1 represses a subset of IFNgamma-inducible STAT1 target genes in a methyltransferase-dependent manner. These genes are also regulated by the STAT1 inhibitor PIAS1 (protein inhibitor of activated STAT1). PIAS1 is arginine methylated by PRMT1 in vitro as well as in vivo upon IFN treatment. Mutational and mass spectrometric analysis of PIAS1 identifies Arg 303 as the single methylation site. Using both methylation-deficient and methylation-mimicking mutants, we find that arginine methylation of PIAS1 is essential for the repressive function of PRMT1 in IFN-dependent transcription and for the recruitment of PIAS1 to STAT1 target gene promoters in the late phase of the IFN response. Methylation-dependent promoter recruitment of PIAS1 results in the release of STAT1 and coincides with the decline of STAT1-activated transcription. Accordingly, knockdown of PRMT1 or PIAS1 enhances the anti-proliferative effect of IFNgamma. Our findings identify PRMT1 as a novel and crucial negative regulator of STAT1 activation that controls PIAS1-mediated repression by arginine methylation.