Accumulating evidence suggests that protein tyrosine phosphorylation-based signaling pathways are under the regulation of reactive oxygen species. Although protein tyrosine phosphatases are directly regulated by reversible oxidation, it is not clear whether protein tyrosine kinases (PTKs) are also directly regulated by reduction/oxidation (redox). In this study we report a mechanism of direct oxidative inactivation specific for the PTKs in the Src and fibroblast growth factor receptor (FGFR) families, key enzymes in mammalian signal transduction. Src is fully active when reduced and retains 8 -25% of the full activity toward various substrates when oxidized. This inactivation is caused by oxidation of a specific cysteine residue (Cys-277), which results in homodimerization of Src linked by a disulfide bridge. Cys-277 is located in the Gly loop in the catalytic domain. This cysteine residue is conserved only in 8 of the >90 PTKs in the human kinome, including 3 of the 10 Src family kinases and all 4 kinases of the FGFR family. FGFR1 is also reversibly regulated by redox because of this cysteine residue, whereas Csk, a PTK that lacks a cysteine residue at the corresponding position, is not similarly regulated. These results demonstrate a mechanism of direct redox regulation conserved in certain specific PTKs. R eactive oxygen species (ROS), such as hydrogen peroxide and superoxide, can alter the function of proteins by oxidizing free sulfhydryl groups to sulfenic, sulfinic, or sulfonic acids (1, 2). Cellular responses to ROS are historically considered a damage-control mechanism to certain pathological situations that lead to oxidative stress. However, recent studies indicate that certain growth factors and cell adhesion also stimulate the production of ROS, which serve as secondary messengers to regulate downstream signaling pathways (3, 4). Numerous protein phosphorylation pathways respond to ROS (5-9), and identifying the proteins and the residues sensitive to oxidation will help elucidate the mechanism of cross-talk between redox and protein tyrosine phosphorylation.The level of protein tyrosine phosphorylation is the function of opposing actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). All PTPs contain a catalytic Cys residue in the active site, and oxidation of this residue leads to the inactivation of the PTP activity (10). This response is recognized as a major mechanism by which ROS regulate the level of protein tyrosine phosphorylation. However, whether PTKs also directly respond to ROS is not established. PTK Src is a key regulator of cell survival, cytoskeleton reorganization, DNA synthesis, and cell division (11,12). A number of studies suggest that Src also plays an important role in cellular response to ROS, because Src specific inhibitors and dominantnegative Src mutants strongly attenuate cellular response to ROS (13-16). However, how ROS regulate Src activity has been controversial, likely reflecting the complexity of Src regulation. Src contains regulatory str...

Protein tyrosine kinases (PTK) are key enzymes of mammalian signal transduction. For the fidelity of signal transduction, each PTK phosphorylates only one or a few proteins on specific Tyr residues. Substrate specificity is thought to be mediated by PTKsubstrate docking interactions and recognition of the phosphorylation site sequence by the kinase active site. However, a substratedocking site has not been determined on any PTK. C-terminal Src kinase (Csk) is a PTK that specifically phosphorylates Src family kinases on a C-terminal Tyr. In this study, by sequence alignment and site-specific mutagenesis, we located a substrate-docking site on Csk. Mutations in the docking site disabled Csk to phosphorylate, regulate, and complex with Src but only moderately affected its general kinase activity. A peptide mimicking the docking site potently inhibited (IC50 ‫؍‬ 21 M) Csk phosphorylation of Src but only moderately inhibited (IC50 ‫؍‬ 422 M) its general kinase activity. Determination of the substrate-docking site provides the structural basis of substrate specificity in Csk and a model for understanding substrate specificity in other PTKs.

In addition to the magnesium ion needed to form the true phosphate-donating substrate (ATP-Mg complex), we have determined that at least one additional Mg2+ ion is essential for the activation of protein tyrosine kinases. This activation was investigated in detail using purified Csk, Src, and the fibroblast growth factor receptor kinase, which led to the following conclusions. (1) The catalytic activity of these kinases is dependent on the Mg2+ concentration present in the assay, approaching saturation at 5-8 mM MgCl2, while ATP was saturated at approximately 1 mM MgCl2. (2) Extrapolation to zero free Mg2+ at a constant ATP-Mg concentration predicts zero activity, suggesting that free magnesium ion in excess of that needed to bind to ATP is essential for the activation of these enzymes. (3) The free magnesium ion activates Csk and Src kinase activity by increasing the Vmax but does not change their apparent Km(ATP-Mg). In contrast, the free magnesium ion activates the fibroblast growth factor receptor kinase activity by increasing its Vmax and decreasing its apparent Km(ATP-Mg). These and previous studies with the insulin receptor tyrosine kinase suggest that receptor-type protein tyrosine kinases respond to the concentration of free Mg2+ differently than soluble protein tyrosine kinases. (4) With the phosphate-accepting substrate as the variable ligand, increases in the concentration of free Mg2+ resulted in increases in the apparent Vmax for all tyrosine kinases examined, but the apparent Km response is dependent on the enzyme and the substrate used. While these studies do not pinpoint a single kinetic mechanism, they do suggest that additional magnesium ion(s) is(are) an essential activator for protein tyrosine kinases in addition to being a part of the ATP-Mg complex. The difference among protein tyrosine kinases in their kinetic response to the additional divalent metal cation and the potential biological significance of such are discussed.

Csk phosphorylates Src family protein tyrosine kinases on a tyrosine residue near their C-terminus and downregulates their activity. We previously observed that this regulation requires a stoichiometric ratio of Csk : Src in a time-independent manner. In this report we examined this unusual kinetic behavior and found it to be caused by Src autophosphorylation. First, pre-incubation of Src with ATP-Mg led to time-dependent autophosphorylation of Src, activation of its kinase activity and loss of its ability to be inactivated by Csk. However, the autophosphorylated Src can still be phosphorylated by Csk. The SH2 binding site for phospho-Tyr of this hyperactive and doubly phosphorylated form of Src is not accessible. Second, dephosphorylation of autophosphorylated Src by protein tyrosine phosphatase 1B allowed Src to be inactivated by Csk. Third, protein tyrosine phosphatase 1B preferentially dephosphorylates the Src autophosphorylation site and allows for Src regulation by Csk. Finally, Yes, another member of the Src family, was also only partially inactivated when a sub-stoichiometric amount of Csk was used. Mutation of the tyrosine autophosphorylation site of Yes to a phenylalanine resulted in a mutant Yes enzyme that can be fully inactivated by a sub-stoichiometric amount of Csk in a time-dependent manner. These results demonstrate that Csk phosphorylation inactivates Src and Yes only when they are not previously autophosphorylated and Src autophosphorylation can block the inactivation by Csk phosphorylation. This conclusion suggests a dynamic model for the regulation of the Src family protein tyrosine kinases, which is discussed in the context of previously reported observations on the regulation of Src family protein tyrosine kinases.

Background: ChatGPT, an artificial intelligence-driven, pretrained, deep learning language model, can generate natural language text in response to a given query. Its rapid growth has led to concerns about ethical use in academia. Problem: The exponential rise in the popularity of ChatGPT, and concerns of academic integrity with its use, has raised concerns among faculty for how to best address this issue. Approach: Faculty should understand the potential benefits and limitations of ChatGPT and create assignments that emphasize self-reflection, critical thinking, problem solving, and independent learning. Students must be taught how to critically evaluate information and how to make informed decisions.Conclusions: ChatGPT has the potential to revolutionize nursing education. However, it is critical for faculty to be familiar with its capabilities and limitations in order to foster effective, yet ethical and responsible utilization, while preparing students for the dynamic, rapidly advancing technological landscape in nursing and health care.

In addition to a magnesium ion needed to form the ATP-Mg complex, we have previously determined that at least one more free Mg 2+ ion is essential for the activation of the protein tyrosine kinase, Csk [

The COOH-terminal Src kinase (Csk) regulates a broad array of cellular processes via the specific phosphorylation and downregulation of Src family protein kinases. While Csk has been a topic for steady-state kinetic studies, the individual steps associated with substrate phosphorylation have not been investigated. To understand active-site phenomena, pre-steady-state and transient-state kinetic methods were applied to develop a catalytic pathway for substrate processing. Rapid quench flow techniques show that the phosphorylation of a substrate peptide, generated from a random library, occurs in two kinetic phases: a rapid, exponential "burst" phase followed by a slow, linear phase. The amplitude of the burst phase increases as a function of enzyme concentration, indicating that the biphasic kinetics are not the result of product inhibition. Analysis of the burst rate as a function of substrate concentration indicates that the phosphoryl transfer step is fast (k3 > or = 140 s(-1) and highly favorable (k3/k-3 > or = 6). The apparent dissociation rate constant for ADP (0.6 s(-1), measured using stopped-flow kinetic methods and a fluorescent trapping agent, mant-ATP, is close to kcat. Since the substrate dissociation constant is high, the release of phosphopeptide is not likely to limit turnover. These findings indicate that Csk rapidly delivers the gamma-phosphate of ATP to the substrate and rapidly releases the phosphoproduct. Overall rate limitation in the steady state is then attributed to the slow, net dissociation of ADP. Viscosometric studies suggest that this final event in the catalytic cycle is coupled with slow conformational changes.

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