Mechanism of activation for Zap-70 catalytic activity.

There is a growing body of evidence, including data from human genetic and T-cell receptor function studies, which implicate a zeta-associated protein of M(r) 70,000 (Zap-70) as a critical protein tyrosine kinase in T-cell activation and development. During T-cell activation, Zap-70 becomes associated via its src homology type 2 (SH2) domains with tyrosine-phosphorylated immune-receptor tyrosine activating motif (ITAM) sequences in the cytoplasmic zeta chain of the T-cell receptor. An intriguing conundrum is how Zap-70 is catalytically activated for downstream phosphorylation events. To address this question, we have used purified Zap-70, tyrosine phosphorylated glutathione S-transferase (GST)-Zeta, and GST-Zeta-1 cytoplasmic domains, and various forms of ITAM-containing peptides to see what effect binding of zeta had upon Zap-70 tyrosine kinase activity. The catalytic activity of Zap-70 with respect to autophosphorylation increased approximately 5-fold in the presence of 125 nM phosphorylated GST-Zeta or GST-Zeta-1 cytoplasmic domain. A 20-fold activity increase was observed for phosphorylation of an exogenous substrate. Both activity increases showed a GST-Zeta concentration dependence. The increase in activity was not produced with nonphosphorylated GST-Zeta, phosphorylated zeta, or phosphorylated ITAM-containing peptides. The increase in Zap-70 activity was SH2 mediated and was inhibited by phenylphosphate, Zap-70 SH2, and an antibody specific for Zap-70 SH2 domains. Since GST-Zeta and GST-Zeta-1 exist as dimers, the data suggest Zap-70 is activated upon binding a dimeric form of phosphorylated zeta and not by peptide fragments containing a single phosphorylated ITAM. Taken together, these data indicate that the catalytic activity of Zap-70 is most likely activated by a trans-phosphorylation mechanism.

Residue 225 determines the Na(+)-induced allosteric regulation of catalytic activity in serine proteases.

Residue 225 in serine proteases is typically Pro or Tyr and specifies an important and unanticipated functional aspect of this class of enzymes. Proteases with Y225, like thrombin, are involved in highly specialized functions like blood coagulation and complement that are exclusively found in vertebrates. In these proteases, the catalytic activity is enhanced allosterically by Na+ binding. Proteases with P225, like trypsin, are typically involved in digestive functions and are also found in organisms as primitive as eubacteria. These proteases have no requirement for Na+ or other monovalent cations. The molecular origin of this physiologically important difference is remarkably simple and is revealed by a comparison of the Na+ binding loop of thrombin with the homologous region of trypsin. The carbonyl O atom of residue 224 makes a key contribution to the coordination shell of the bound Na+ in thrombin, but is oriented in a manner incompatible with Na+ binding in trypsin because of constraints imposed by P225 on the protein backbone. Pro at position 225 is therefore incompatible with Na+ binding and is a direct predictor of the lack of allosteric regulation in serine proteases. To directly test this hypothesis, we have engineered the thrombin mutant Y225P. This mutant has lost the ability to bind Na+ and behaves like the allosteric slow (Na(+)-free) form. The Na(+)-induced allosteric regulation also bears on the molecular evolution of serine proteases. A strong correlation exists between residue 225 and the codon used for the active site S195. Proteases with P225 typically use a TCN codon for S195, whereas proteases with Y225 use an AGY codon. It is proposed that serine proteases evolved from two main lineages: (i) TCN/P225 with a trypsin-like ancestor and (ii) AGY/Y225 with a thrombin-like ancestor. We predict that the Na(+)-induced allosteric regulation of catalytic activity can be introduced in the TCN/P225 lineage using the P225Y replacement.

Catalytic activity of the mouse guanine nucleotide exchanger mSOS is activated by Fyn tyrosine protein kinase and the T-cell antigen receptor in T cells.

mSOS, a guanine nucleotide exchange factor, is a positive regulator of Ras. Fyn tyrosine protein kinase is a potential mediator in T-cell antigen receptor signal transduction in subsets of T cells. We investigated the functional and physical interaction between mSOS and Fyn in T-cell hybridoma cells. Stimulation of the T-cell antigen receptor induced the activation of guanine nucleotide exchange activity in mSOS immunoprecipitates. Overexpression of Fyn mutants with an activated kinase mutation and with a Src homology 2 deletion mutation resulted in a stimulation and suppression of the mSOS activity, respectively. The complex formations of Fyn-Shc, Shc-Grb2, and Grb2-mSOS were detected in the activated Fyn-transformed cells, whereas the SH2 deletion mutant of Fyn failed to form a complex with mSOS. Moreover, tyrosine phosphorylation of Shc was induced by the overexpression of the activated Fyn. These findings support the idea that Fyn activates the activity of mSOS bound to Grb2 through tyrosine phosphorylation of Shc. Unlike the current prevailing model, Fyn-induced activation of Ras might involve the stimulation of the catalytic guanine nucleotide exchange activity of mSOS.

A novel laponite clay-based Fe nanocomposite and its photo-catalytic activity in photo-assisted degradation of Orange II

A novel laponite RD clay-based Fe nanocomposite (Fe-Lap-RD) has been successfully synthesized through a reaction between a solution of iron salt and an aqueous dispersion of laponite RD clay. The X-ray diffraction (XRD) results reveal that the Fe-Lap-RD mainly consists of Fe2O3 (maghemite) and Fe2Si4O10(OH)2 (iron silicate hydroxide), which have tetragonal and monoclinic structures, respectively, and has a high specific surface area as well as a high pore volume. The photo-catalytic activity of the Fe-Lap-RD was examined in the photo-assisted degradation of an organic azo dye Orange II. It was found that the mineralization of Orange 11 undergoes a slower kinetics than discoloration, and 70% total organic carbon (TOC) of 0.2 mM Orange 11 can be removed in 90 min, implying that the Fe-Lap-RD exhibited a high photo-catalytic activity in the presence of H2O2 and UV light (254 nm) in the photo-assisted degradation of Orange II. In addition, our experiments also illustrate that the Fe-Lap-RD has a long-term stability but is of low cost. This study illustrates the possibility of photo-assisted degradation of organic compounds without the requirements to remove the Fe ions after reaction. Two possible catalytic reaction mechanisms are also proposed. (C) 2003 Elsevier Science Ltd. All rights reserved.