Biomedical subjects

Michael Freeley

Publications and source records attributed to Michael Freeley.

5 recordsLinked to original sources

PubMed2006

Comment on "PDK1 nucleates T cell receptor-induced signaling complex for NF-kappaB activation".

We observe that protein kinase C (PKC) is phosphorylated on the activation loop at threonine 538 (Thr-538) before T cell activation. Our results are inconsistent with the conclusions of Lee et al. (Reports, 1 April 2005, p. 114) that the Thr-538 phosphorylation of PKC is regulated by T cell receptor activation. Other mechanisms, such as autophosphorylation of Thr-219, might orchestrate the cellular function of PKC in T cells.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2006

The hepatitis C envelope 2 protein inhibits LFA-1-transduced protein kinase C signaling for T-lymphocyte migration.

BACKGROUND & AIMS: The ability of viruses to escape the host immune response represents a globally important problem related to a wide variety of pathogens. Hepatitis C is one of the major causes of liver disease worldwide. Clearance rates of this virus are low, and this condition normally involves a chronic inflammatory process. This raises a possibility that the virus may have developed mechanisms enabling it to evade T-cell-mediated immune surveillance. The aim of this study was to investigate the effect of the hepatitis C envelope protein E2 on LFA-1-stimulated T-cell migration and macrophage inflammatory protein (MIP-1alpha, MIP-1beta) secretion. METHODS: T cells were stimulated through the leukocyte function-associated molecule-1 (LFA-1) receptor by incubating with either intracellular adhesion molecule 1 (ICAM-1)-Fc fusion protein or anti-LFA-1 immobilized on 8-well chamber slides. Subcellular localization of protein kinase C (PKC)-beta, CD81, and LFA-1 was determined by immunofluorescence analysis. Lipid raft formation was assessed using the Cellomics Kineticscan reader. MIP-1alpha and MIP-1beta levels were detected by enzyme-linked immunosorbent assay. RESULTS: We report that the hepatitis C envelope protein E2 can dramatically inhibit T-lymphocyte motility and chemokine release induced via LFA-1 integrin ligation. We have demonstrated a novel T-lymphocyte-directed viral inhibitory mechanism involving the PKC-beta enzyme as a definitive intracellular target. E2-CD81 interaction stimulates translocation of PKC-beta to lipid rafts, thereby preventing its association with the centrosome and microtubule cytoskeleton, which is crucial to the process of T-cell migration. CONCLUSIONS: These studies identify a mechanism whereby the hepatitis C virus can evade the host immune response by inhibition of T-cell migration.

Cell Movement↗

PubMed2005

Stimulus-induced phosphorylation of PKC theta at the C-terminal hydrophobic-motif in human T lymphocytes.

Protein kinase C (PKC) is a family of serine/threonine kinases whose activity is controlled, in part, by phosphorylation on three conserved residues that are located on the catalytic domain of the enzyme, known as the activation-loop, the turn-motif, and the C-terminal hydrophobic-motif sites. Using a panel of phospho-specific antibodies, we have determined that PKC beta(I) and delta are constitutively phosphorylated on all three sites in unstimulated and activated T cells. Although PKC theta is constitutively phosphorylated at the activation-loop and turn-motif sites in T cells, PMA or anti-CD3/CD28 stimulation results in an increase in phosphorylation at the hydrophobic-motif (Ser695), an event that coincides with translocation of the enzyme from the cytosol/cytoskeleton to the membrane. Studies on the stimulus-induced phosphorylation of PKC theta demonstrate that an upstream kinase activity involving a conventional PKC isoform(s) and the PI3-kinase pathway, rather than autophosphorylation or the rapamycin-sensitive mTOR pathway, regulates this site in T lymphocytes. However, hydrophobic-motif phosphorylation does not appear to control membrane translocation, suggesting that this site may control other aspects of PKC theta signalling.

Amino Acid Motifs↗

PubMed2005

Microdamage: a cell transducing mechanism based on ruptured osteocyte processes.

As a result of underlying pathological diseases, such as osteoporosis, osteopenia, or due to altered loading after joint replacements, bones become more susceptible to microdamage accumulation than those of normal human beings, as are those of athletes who undertake strenuous exercise [Stromsoe, 2004. Fracture fixation problems in osteoporosis. Injury 35, 107-113]. Experimental evidence has linked bone adaptation to microdamage, and to increased cell activity. In this work, we investigated whether microcrack detection is related to rupturing of the cellular material itself due to crack face displacements. Using specific cell staining techniques, it was confirmed that relative crack displacements are capable of tearing cell processes between neighbouring osteocytes. No ruptured cell processes were found near the crack tip where the displacements are less. Rupturing of cell processes due to crack opening and shear displacement is a feasible new mechanism by which bone can detect and estimate the size of a microcrack. Ruptured cell processes may directly secrete passive and active components in the extracellular matrix, triggering a repair response.

Adaptation, Physiological↗

PubMed2005

CD44 cross-linking induces protein kinase C-regulated migration of human T lymphocytes.

The cell surface receptor CD44 is widely implicated in leukocyte migration to inflammatory sites. In this study, the responses of human T cells following cross-linking of CD44 were examined. We demonstrate that engagement of CD44 using immobilized mAbs or hyaluronan-enriched extracellular matrix lattices induces active migration in T lymphocytes accompanied by cycles of cytoskeletal rearrangement and cell polarization. We have investigated the functional impact and subcellular localization of protein kinase C (PKC) isoenzymes, beta and delta, previously shown by our group to be involved in active T cell locomotion induced by leukocyte function-associated antigen-1 (LFA-1) integrin receptors. PKCbeta was associated with the centrosome and the microtubule-rich tail of the polarized cell and PKCdelta was predominantly located about the region of the microtubule organizing center. A selective pharmacological inhibitor of classical PKC isoforms, Go6976, suppressed lymphocyte polarization and migration following CD44 ligation. Selective targeting of PKCdelta using the pharmacological inhibitor rottlerin or a pseudosubstrate-blocking peptide reduced CD44-activated cell migration but did not completely ablate it. Our data demonstrate that ligation of CD44 induces phenotypic changes, cytoskeletal rearrangements and redistribution of PKC isoforms beta and delta, resulting in cell migration, as previously described for the cell surface receptor, LFA-1. This suggests potential convergence of intracellular signaling pathways induced via CD44 and LFA-1 integrin.

Cell Movement↗