Direct interaction in T-cells between thetaPKC and the tyrosine kinase p59fyn.

The protein kinase C (PKC) family has been clearly implicated in T-cell activation as have several nonreceptor protein-tyrosine kinases associated with the T-cell receptor, including p59fyn. This report demonstrates that thetaPKC and p59fyn specifically interact in vitro, in the yeast two-hybrid system, and in T-cells. Further indications of direct interaction are that p59fyn potentiates thetaPKC catalytic activity and that thetaPKC is a substrate for tyrosine phosphorylation by p59fyn. This interaction may account for the localization of thetaPKC following T-cell activation, pharmacological disruption of which results in specific cell-signaling defects. The demonstration of a physical interaction between a PKC and a protein-tyrosine kinase expands the class of PKC-anchoring proteins (receptors for activated C kinases (RACKs)) and demonstrates a direct connection between these two major T-cell-signaling pathways.

Glubodies: randomized libraries of glutathione transferase enzymes.

BACKGROUND: The immunoglobulin framework has been mutagenized to engineer recombinant libraries of proteins as potential diagnostics and novel catalysts, although the often shallow binding cleft may limit the utility of this framework for binding diverse small organic molecules. By contrast, the glutathione S-transferase (GST) family of enzymes contains a deep binding cleft, which has evolved to accommodate a broad range of hydrophobic xenobiotics. We set out to determine whether GST molecules with novel ligand-binding characteristics could be produced by random mutagenesis of segments of the binding cleft. RESULTS: We have identified two ligand-recognition segments (LRSs) in human GST P1, which are near the active site in the folded protein, but have characteristics indicating that the integrity of their sequence is not essential for the overall structure or activity of the protein. Libraries of GST P1-derived proteins were produced by substituting randomized sequences for an LRS or inserting random sequences into an LRS. The recombinant proteins in the libraries, collectively designated as 'glubodies,' generally retain enzymatic activity but differ markedly both from each other and from the parent enzyme in sensitivity to inhibition by diverse small organic compounds. In some instances, a glubody is inhibited by completely novel structures. CONCLUSIONS: We have shown that a non-antibody framework can be used to create large libraries of proteins with a wide range of binding specificities for small organic molecules. The glubodies provide a rich source of data for correlating the structural and functional features of proteins relevant to ligand binding. The criteria applied for identifying an LRS in GST P1 are generally applicable to other protein frameworks.

Mutagenesis using trinucleotide beta-cyanoethyl phosphoramidites.

There is no easy way to selectively introduce mixtures of codon triplets into mutagenesis libraries. Solid-phase-supported DNA synthesis using successive coupling of mixtures of mononucleotides can be made to supply 32 codons, which gives redundancies in coding for 20 natural amino acids, as well as an often unwanted stop codon. Resin-splitting methods have been described, but the representation of all permutations is limited by mechanical factors for a large library, and the method is experimentally cumbersome. To demonstrate a third, improved method, the 3'-cyanoethyl phosphoramidite codon triplets dATA, dCTT, dATC, dATG and dAGC were made by solution-phase methods, with protecting groups fully compatible with modern automated phosphoramidite DNA synthesis chemistry. The reagents were then used to synthesize a 54-mer DNA fragment, wherein 15 internal base pairs were randomized by coupling a mixture of the five codons five times. The fragment was amplified as a cDNA pool, which was subcloned into a phagemid vector, and 16 randomly selected recombinants from this mini-library were sequenced. These clones showed random incorporation of the proper transcribed codon sequences at the correct location. Other functional tests involving the trinucleotide phosphoramidites showed modest (ca. 70%) coupling efficiencies and structural integrity of the DNA produced.