No abstract

Insulin has pleiotropic effects on the regulation of cell physiology through binding to its receptor. The wide variety of tyrosine phosphorylation motifs of insulin receptor substrate 1 (IRS-1), a substrate for the activated insulin receptor tyrosine kinase, may account for the multiple functions of insulin. Recent studies have shown that activation of the insulin receptor leads to the regulation of focal adhesion proteins, such as a dephosphorylation of focal adhesion kinase (pp125 FAK ). We show here that C-terminal Src kinase (Csk), which phosphorylates C-terminal tyrosine residues of Src family protein tyrosine kinases and suppresses their kinase activities, is involved in this insulin-stimulated dephosphorylation of focal adhesion proteins. We demonstrated that the overexpression of Csk enhanced and prolonged the insulin-induced dephosphorylation of pp125 FAK . Another focal adhesion protein, paxillin, was also dephosphorylated upon insulin stimulation, and a kinase-negative mutant of Csk was able to inhibit the insulin-induced dephosphorylation of pp125 FAK and paxillin. Although we have shown that the Csk Src homology 2 domain can bind to several tyrosine-phosphorylated proteins, including pp125FAK and paxillin, a majority of protein which bound to Csk was IRS-1 when cells were stimulated by insulin. Our data also indicated that tyrosine phosphorylation levels of IRS-1 appear to be paralleled by the dephosphorylation of the focal adhesion proteins. We therefore propose that the kinase activity of Csk, through the insulin-induced complex formation of Csk with IRS-1, is involved in insulin's regulation of the phosphorylation levels of the focal adhesion proteins, possibly through inactivation of the kinase activity of c-Src family kinases.Among the earliest cellular responses following insulin stimulation are the tyrosine phosphorylations of insulin receptor (IR) ␤ subunit as well as IR substrate 1 (IRS-1) (pp185) by the activated IR kinase activity (22, 23, 52, 64; for a review, see reference 62). IRS-1, a cytoplasmic protein found in most cells and tissues, contains more than 20 potential tyrosine phosphorylation sites; several of these sites have been shown to bind specifically to each of the Src homology 2 (SH2) domains of various signaling proteins such as phosphatidylinositol (PI) 3Ј-kinase, Ash/Grb2, Syp, and Nck (3,4,26,27,29,50,52,58). It is possible that the divergent structures and functions of the SH2 proteins which bind to IRS-1 mediate the pleiotropic responses of insulin; tyrosine phosphorylation of IRS-1 may act as a switch to control the interaction of IRS-1 with these signaling proteins (52). In addition to IRS-1, another substrate, pp190 (IRS-2), which was found in IRS-1-deficient mice and can substitute, at least in part, for the function of IRS-1, has been reported. IRS-2 also binds to PI 3Ј-kinase (2,53,55,59) and Ash/Grb2 (53, 59), suggesting that it too serves as an SH2-docking protein.

In cynomolgus monkeys, twice daily subcutaneous injections of recombinant human interleukin-6 (rhIL-6) at doses of 5 to 80 micrograms/kg/d for 14 consecutive days caused dose-dependent increases in platelet count, usually continuing for more than 1 week after cessation of the injections. The count reached a level approximately twofold or more above the preinjection level even at 5 micrograms/kg/d, and at doses of more than 20 micrograms/kg/d, the increase became biphasic with a higher second peak 3 days after cessation of the injections. Morphologic analysis of the bone marrow after the 7 day- injections with 80 micrograms/kg/d revealed a marked increment in size of megakaryocytes compared with control, indicating the promotion of megakaryocyte maturation. Other changes attributable to the rhIL-6 treatment include dose-dependent loss of body weight, anemia, neutrophilia and monocytosis, elevation of serum C-reactive protein and alpha-1 acid glycoprotein levels, and decrease of serum albumin; all of which returned to normal within 1 week after cessation of the injections and were tolerable at doses of less than 10 micrograms/kg/d. These findings suggest that rhIL-6 may be an effective strategy for the treatment of thrombocytopenia.

In ribosomal translation, the accommodation of aminoacyl-tRNAs into the ribosome is mediated by elongation factor thermo unstable (EF-Tu). The structures of proteinogenic aminoacyl-tRNAs (pAA-tRNAs) are fine-tuned to have uniform binding affinities to EF-Tu in order that all proteinogenic amino acids can be incorporated into the nascent peptide chain with similar efficiencies. Although genetic code reprogramming has enabled the incorporation of non-proteinogenic amino acids (npAAs) into the nascent peptide chain, the incorporation of some npAAs, such as N-methyl-amino acids (MeAAs), is less efficient, especially when MeAAs frequently and/or consecutively appear in a peptide sequence. Such poor incorporation efficiencies can be attributed to inadequate affinities of MeAA-tRNAs to EF-Tu. Taking advantage of flexizymes, here we have experimentally verified that the affinities of MeAA-tRNAs to EF-Tu are indeed weaker than those of pAA-tRNAs. Since the T-stem of tRNA plays a major role in interacting with EF-Tu, we have engineered the T-stem sequence to tune the affinity of MeAA-tRNAs to EF-Tu. The uniform affinity-tuning of the individual pairs has successfully enhanced the incorporation of MeAAs, achieving the incorporation of nine distinct MeAAs into both linear and thioether-macrocyclic peptide scaffolds.

It has been well established that the ribosome can accept various nucleophiles on the Xacyl-tRNA in the A site during elongation, where X can be amino, N-alkyl-amino, hydroxy, and thiol groups. However, it remains elusive that the ribosome is able to accept an electrophile in the P site other than the carboxyl group during elongation. Here we report ribosomal formation of a thioamide bond in the mRNA-dependent polypeptide synthesis. In this study, amino­(carbothio)­acyl-tRNA was prepared by flexizyme and used for the expression of peptides containing a thioamide bond in the nascent peptide chain. We give strong evidence that the thioamide-peptide was formed but accompanied by the amide counterpart due to rapid carbo­(S-to-O) exchange during the preparation of amino­(carbothio)­acyl-tRNA. We also demonstrate the ribosomal formation of thioamide and N-methyl-thioamide bonds in linear as well as macrocyclic peptide scaffolds in the mRNA-dependent manner, showing its potential for applications in peptide-based drug discovery and studying peptide/protein structure and function.