The proximal signaling network of the BCR-ABL1 oncogene shows a modular organization.

BCR-ABL1 is a fusion tyrosine kinase, which causes multiple types of leukemia. We used an integrated proteomic approach that includes label-free quantitative protein complex and phosphorylation profiling by mass spectrometry to systematically characterize the proximal signaling network of this oncogenic kinase. The proximal BCR-ABL1 signaling network shows a modular and layered organization with an inner core of three leukemia transformation-relevant adaptor protein complexes (Grb2/Gab2/Shc1 complex, CrkI complex and Dok1/Dok2 complex). We introduced an 'interaction directionality' analysis, which annotates static protein networks with information on the directionality of phosphorylation-dependent interactions. In this analysis, the observed network structure was consistent with a step-wise phosphorylation-dependent assembly of the Grb2/Gab2/Shc1 and the Dok1/Dok2 complexes on the BCR-ABL1 core. The CrkI complex demonstrated a different directionality, which supports a candidate assembly on the Nedd9 (Hef1, CasL) scaffold. As adaptor protein family members can compensate for each other in leukemic transformation, we compared members of the Dok and Crk protein families and found both overlapping and differential binding patterns. We identified an additional level of regulation for the CrkII protein via binding to 14-3-3 proteins, which was independent from its inhibitory phosphorylation. We also identified novel components of the inner core complexes, including the kinases Pragmin (Sgk223) and Lrrk1 (Lrrk2 paralog). Pragmin was found as a component of the CrkI complex and is a potential link between BCR-ABL1/CrkI and RhoA signaling. Lrrk1 is an unusual kinase with a GTPase domain. We detected Lrrk1 as a component of the Grb2/Gab2/Shc1 complex and found that it functionally interacts with the regulator of small GTPases Arap1 (Centd2) and possibly participates in the mitogen-activated protein kinase response to cellular stresses. This modular and phosphorylation-driven interaction network provides a framework for the integration of pleiotropic signaling effects of BCR-ABL1 toward leukemic transformation.

Interaction between yeast RNA polymerase III and transcription factor TFIIIC via ABC10alpha and tau131 subunits.

Yeast TFIIIC mediates transcription of class III genes by promoting the assembly of a stable TFIIIB-DNA complex that is sufficient for RNA polymerase III recruitment and function. Unexpectedly, we found an interaction in vivo and in vitro between the TFIIIB-recruiting subunit of TFIIIC, tau131, and ABC10alpha, a small essential subunit common to the three forms of nuclear RNA polymerases. This interaction was mapped to the C-terminal region of ABC10alpha. A thermosensitive mutation in the C terminus region of ABC10alpha (rpc10-30) was found to be selectively suppressed by overexpression of a mutant form of tau131 (tau131-DeltaTPR2) that lacks the second TPR repeat. Remarkably, the rpc10-30 mutation weakened the ABC10alpha-tau131 interaction, and the suppressive mutation, tau131-DeltaTPR2 increased the interaction between the two proteins in the two-hybrid assay. These results point to the potential importance of a functional contact between TFIIIC and RNA polymerase III.

Functional characterization of ABC10alpha, an essential polypeptide shared by all three forms of eukaryotic DNA-dependent RNA polymerases.

ABC10alpha is a small polypeptide shared by the three yeast RNA polymerases. Homologous polypeptides in higher eukaryotes have a zinc-binding CX(2)C. CX(2)C motif and a conserved basic C-terminal end. These features are also found in archaeal gene products that may encode an RNA polymerase subunit. The CX(2)C. CX(2)C motif is partly dispensable, since only its first cysteine is essential for growth, whereas the basic C-terminal end is critical in vivo. A mutant in the latter domain has an RNA polymerase III-specific defect and, in vitro, impairs RNA polymerase III assembly. Polymerase activity was, however, not affected in various faithful transcription assays. The mutant is suppressed by a high gene dosage of the second largest subunit of RNA polymerase III, whereas the homologous subunits of RNA polymerase I and II have aggravating effects. In a two-hybrid assay, ABC10alpha binds to the C-terminal half of the second largest subunit of RNA polymerase I, in a way that requires the integrity of the CX(2)C. CX(2)C motif. Thus, ABC10alpha appears to interact directly with the second largest subunit during polymerase assembly. This interaction is presumably a major rate-limiting step in assembly, since diploid cells containing only one functional gene copy for ABC10alpha have a partial growth defect.

A protein-protein interaction map of yeast RNA polymerase III.

The structure of the yeast RNA polymerase (pol) III was investigated by exhaustive two-hybrid screening using a library of random genomic fragments fused to the Gal4 activation domain. This procedure allowed us to identify contacts between individual polypeptides, localize the contact domains, and deduce a protein-protein interaction map of the multisubunit enzyme. In all but one case, pol III subunits were able to interact in vivo with one or sometimes two partner subunits of the enzyme or with subunits of TFIIIC. Four subunits that are common to pol I, II, and III (ABC27, ABC14.5, ABC10alpha, and ABC10beta), two that are common to pol I and III (AC40 and AC19), and one pol III-specific subunit (C11) can associate with defined regions of the two large subunits. These regions overlapped with highly conserved domains. C53, a pol III-specific subunit, interacted with a 37-kDa polypeptide that copurifies with the enzyme and therefore appears to be a unique pol III subunit (C37). Together with parallel interaction studies based on dosage-dependent suppression of conditional mutants, our data suggest a model of the pol III preinitiation complex.

Chromatin structure of the Saccharomyces cerevisiae DNA topoisomerase I promoter in different growth phases.

We have determined the chromatin organization of the Saccharomyces cerevisiae DNA topoisomerase I promoter. Three nucleosomal core particles have been mapped at nucleotide level over the promoter region, encompassing the presumptive TATA sequence and the two RNA initiation sites; the most upstream nucleosome particle forms on to a 29 bp-long poly(dA-dT) element. This simple organization remains constant throughout both the logarithmic and the linear phase of growth, with the exception of an increased accessibility to micrococcal nuclease of the nucleosome covering the TATA box and the RNA initiation sites during the diauxic shift (the switching from the fermentative to the respiratory metabolism) in parallel with an increase of the DNA topoisomerase I mRNA. In addition, a strong disorganization of the bulk chromatin structure in the late stationary phase is also reported.