We have previously described a strategy for detecting protein protein interactions based on protein interaction assisted folding of rationally designed fragments of enzymes. We call this strategy the protein fragment complementation assay (PCA). Here we describe PCAs based on the enzyme TEM-1 beta-lactamase (EC: 3.5.2.6), which include simple colorimetric in vitro assays using the cephalosporin nitrocefin and assays in intact cells using the fluorescent substrate CCF2/AM (ref. 6). Constitutive protein protein interactions of the GCN4 leucine zippers and of apoptotic proteins Bcl2 and Bad, and the homodimerization of Smad3, were tested in an in vitro assay using cell lysates. With the same in vitro assay, we also demonstrate interactions of protein kinase PKB with substrate Bad. The in vitro assay is facile and amenable to high-throughput modes of screening with signal-to-background ratios in the range of 10:1 to 250:1, which is superior to other PCAs developed to date. Furthermore, we show that the in vitro assay can be used for quantitative analysis of a small molecule induced protein interaction, the rapamycin-induced interaction of FKBP and yeast FRB (the FKBP-rapamycin binding domain of TOR (target of rapamycin)). The assay reproduces the known dissociation constant and number of sites for this interaction. The combination of in vitro colorimetric and in vivo fluorescence assays of beta-lactamase in mammalian cells suggests a wide variety of sensitive and high-throughput large-scale applications, including in vitro protein array analysis of protein protein or enzyme protein interactions and in vivo applications such as clonal selection for cells expressing interacting protein partners.
Quaking viable (Qk(v)) mice have developmental defects that result in their characteristic tremor. The quaking (Qk) locus expresses alternatively spliced RNA-binding proteins belonging to the STAR family. To characterize the RNA binding specificity of the QKI proteins, we selected for RNA species that bound QKI from random pools of RNAs and defined the QKI response element (QRE) as a bipartite consensus sequence NACUAAY-N(1-20)-UAAY. A bioinformatic analysis using the QRE identified the three known RNA targets of QKI and 1,430 new putative mRNA targets, of which 23 were validated in vivo. A large proportion of the mRNAs are implicated in development and cell differentiation, as predicted from the phenotype of the Qk(v) mice. In addition, 24% are implicated in cell growth and/or maintenance, suggesting a role for QKI in cancer.
The quaking (Qk) locus expresses a family of RNA binding proteins, and the expression of several alternatively spliced isoforms coincides with the development of oligodendrocytes and the onset of myelination. Quaking viable (Qk(v)) mice harboring an autosomal recessive mutation in this locus have uncompacted myelin in the central nervous system owing to the inability of oligodendrocytes to properly mature. Here we show that the expression of two QKI isoforms, absent from oligodendrocytes of Qk(v) mice, induces cell cycle arrest of primary rat oligodendrocyte progenitor cells and differentiation into oligodendrocytes. Injection of retroviruses expressing QKI into the telencephalon of mouse embryos induced differentiation and migration of multipotential neural progenitor cells into mature oligodendrocytes localized in the corpus callosum. The mRNA encoding the cyclin-dependent kinase (CDK)-inhibitor p27(Kip1) was bound and stabilized by QKI, leading to an increased accumulation of p27(Kip1) protein in oligodendrocytes. Our findings demonstrate that QKI is upstream of p27(Kip1) during oligodendrocyte differentiation.
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