Biomedical subjects

Avery August

Publications and source records attributed to Avery August.

23 records · Page 2Linked to original sources

PubMed2002

The proline rich region of the Tec homology domain of ITK regulates its activity.

Inducible T-cell kinase (ITK) is a member of the Tec family of tyrosine kinases that are involved in signals emanating from cytokine receptors, antigen receptors and other lymphoid cell surface receptors. Stimulation of tyrosine phosphorylation and activation of ITK by the T-cell antigen receptor, CD28 and CD2 requires the presence of the Src family kinase Lck in T-cells. We have previously demonstrated that the activation of ITK by Src family kinases uses a phosphatidylinositol 3-kinase pathway, which recruits ITK to the membrane via its pleckstrin homology (PH) domain where it is acted upon by Src. We have further explored the mechanism of this requirement for Src family kinases in the activation of ITK. We found that deletion of the proline rich sequence found in the Tec homology domain of ITK results in reduced basal activity of ITK approximately 50%. These differences in the basal activity of ITK were observed when the PH domain was deleted or when the kinase was membrane targeted. Furthermore, this deletion reduces the ability of the Src family kinase Lck to activate ITK, as well as to induce the ITK mediated tyrosine phosphorylation of its substrate PLCgamma1. By contrast, deletion of the SH3 domain of ITK resulted in a two-fold increase in the basal activity of ITK, and allowed this mutant to have an enhanced response to Lck. These results suggest that the proline rich region within the Tec homology domain of ITK regulates its basal activity and its response to Src family kinase signals.

Blotting, Western↗

PubMed2002

A p130Cas tyrosine phosphorylated substrate domain decoy disrupts v-crk signaling.

BACKGROUND: The adaptor protein p130Cas (Cas) has been shown to be involved in different cellular processes including cell adhesion, migration and transformation. This protein has a substrate domain with up to 15 tyrosines that are potential kinase substrates, able to serve as docking sites for proteins with SH2 or PTB domains. Cas interacts with focal adhesion plaques and is phosphorylated by the tyrosine kinases FAK and Src. A number of effector molecules have been shown to interact with Cas and play a role in its function, including c-crk and v-crk, two adaptor proteins involved in intracellular signaling. Cas function is dependent on tyrosine phosphorylation of its substrate domain, suggesting that tyrosine phosphorylation of Cas in part regulates its control of adhesion and migration. To determine whether the substrate domain alone when tyrosine phosphorylated could signal, we have constructed a chimeric Cas molecule that is phosphorylated independently of upstream signals. RESULTS: We found that a tyrosine phosphorylated Cas substrate domain acts as a dominant negative mutant by blocking Cas-mediated signaling events, including JNK activation by the oncogene v-crk in transient and stable lines and v-crk transformation. This block was the result of competition for binding partners as the chimera competed for binding to endogenous c-crk and exogenously expressed v-crk. CONCLUSION: Our approach suggests a novel method to study adaptor proteins that require phosphorylation, and indicates that mere tyrosine phosphorylation of the substrate domain of Cas is not sufficient for its function.

Binding, Competitive↗

PubMed2002

Recruitment of phosphatidylinositol 3-kinase to CD28 inhibits HIV transcription by a Tat-dependent mechanism.

Activation through the TCR and the costimulatory molecule CD28 influences the susceptibility of T cells to HIV-1 infection and regulates proviral gene expression. Signaling events initiated by CD28 that directly impact HIV-1 transcription have not been fully characterized. T cell lines expressing CD8alpha/28 chimeric receptors containing a mutation in tyrosine 173 to phenylalanine, which inhibits the recruitment of phosphatidylinositol 3-kinase (PI3K) to CD28, expressed higher levels of HIV-1 following T cell activation. Whereas constitutively active PI3K decreased provirus transcription, inhibiting endogenous PI3K with specific inhibitors or by overexpressing PTEN phosphatase enhanced HIV-1 expression. PI3K-dependent inhibition required the viral Tat protein and a trans activation response region element. Tat pull-down and coimmunoprecipitation experiments indicate that PI3K affects the formation of the Tat-associated kinase trans-activating complex. These studies demonstrate that PI3K negatively impacts HIV-1 transcription and that Tat activity is sensitive to T cell signaling events.

Animals↗

PubMed2002

Opposing roles of serine/threonine kinases MEKK1 and LOK in regulating the CD28 responsive element in T-cells.

T-cell activation requires signals from both the T-cell receptor (TcR) and other co-stimulatory molecules such as CD28. TcR- and CD28-mediated signals are integrated during T-cell activation resulting in the expression of cytokine genes such as interleukin-2 (IL-2). An enhancer element (CD28RE) of the IL-2 gene specifically responsive to CD28 signals has been previously identified and characterized. This response element and an adjacent Activated Protein-1 (nuclear factor-interleukin-2B) site together (RE/AP1) were shown to complex with c-rel, AP-1 and other factors. However, details of the signal transduction pathways leading from CD28 to the composite response element remain poorly understood. We present data showing that overexpression of the serine threonine kinase, mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase kinase-1 (MEKK1), but not nuclear factor-kappa B inducing kinase, or MAP kinase/ERK kinase-1 (MEK1), can significantly increase the level of CD28RE/AP1-driven luciferase (Luc) reporter gene expression in Jurkat E6-1 cells. A MEKK1 dominant negative mutant blocked such activation induced by stimulation with Raji B cells and the superantigen staphylococcus enterotoxin E (SEE), as well as via CD3/CD28. Mutations in either site of the RE/AP1 element abolished MEKK1-induced Luc expression. Calcineurin inhibitors, CsA and FK520, or inhibitors of p38 kinase (SB 203580), or MEK1 (PD 098059), did not affect MEKK1-induced reporter activation. These results directly implicate MEKK1 in the CD28 signalling pathway that activates the CD28 response element. Co-expression of the lymphocyte-oriented kinase (LOK) kinase attenuated Raji/SEE-induced IL-2 production in Jurkat cells, as well as MEKK1 and Raji/SEE-induced reporter gene activation. These data suggest that MEKK1 and LOK may have opposing roles in regulating the CD28RE/AP1 element.

CD28 Antigens↗

PubMed2002

The Tec family of tyrosine kinases in T cells, amplifiers of T cell receptor signals.

ITK and Rlk/Txk are the predominant Tec family of tyrosine kinases expressed in T cells, and are involved in T cell antigen receptor mediated activation of T cells. These kinases require prior activation of Lck, Zap-70 and PI3-kinase for efficient activation. They share major substrates with both Lck and Zap-70, however the pathways they regulate are unclear. Recent evidence suggests that these kinases may not activate unique pathways, but instead serve as amplifiers for the upstream kinases Lck and Zap-70. This review will discuss the evidence for this view.

Enzyme Activation↗