Ailsa G. Harpur

Kinase-negative mutants of JAK1 can sustain interferon-gamma-inducible gene expression but not an antiviral state.

The receptor‐associated protein tyrosine kinases JAK1 and JAK2 are both required for the interferon (IFN)‐gamma response. The effects of expressing kinase‐negative JAK mutant proteins on signal transduction in response to IFN‐gamma in wild‐type cells and in mutant cells lacking either JAK1 or JAK2 have been analysed. In cells lacking endogenous JAK1 the expression of a transfected kinase‐negative JAK1 can sustain substantial IFN‐gamma‐inducible gene expression, consistent with a structural as well as an enzymic role for JAK1. Kinase‐negative JAK2, expressed in cells lacking endogenous JAK2, cannot sustain IFN‐gamma‐inducible gene expression, despite low level activation of STAT1 DNA binding activity. When expressed in wild‐type cells, kinase‐negative JAK2 acts as a dominant‐negative inhibitor of the IFN‐gamma response. Further analysis of the JAK/STAT pathway suggests a model for the IFN‐gamma response in which the initial phosphorylation of JAK1 and JAK2 is mediated by JAK2, whereas phosphorylation of the IFN‐gamma receptor is normally carried out by JAK1. The efficient phosphorylation of STAT 1 in the receptor‐JAK complex may again depend on JAK2. Interestingly, a JAK1‐dependent signal, in addition to STAT1 activation, appears to be required for the expression of the antiviral state.

Imaging FRET between spectrally similar GFP molecules in single cells

Fluorescence resonance energy transfer (FRET) detection in fusion constructs consisting of green fluorescent protein (GFP) variants linked by a sequence that changes conformation upon modification by enzymes or binding of ligands has enabled detection of physiological processes such as Ca2+ ion release, and protease and kinase activity. Current FRET microscopy techniques are limited to the use of spectrally distinct GFPs such as blue or cyan donors in combination with green or yellow acceptors. The blue or cyan GFPs have the disadvantages of less brightness and of autofluorescence. Here a FRET imaging method is presented that circumvents the need for spectral separation of the GFPs by determination of the fluorescence lifetime of the combined donor/acceptor emission by fluorescence lifetime imaging microscopy (FLIM). This technique gives a sensitive, reproducible, and intrinsically calibrated FRET measurement that can be used with the spectrally similar and bright yellow and green fluorescent proteins (EYFP/EGFP), a pair previously unusable for FRET applications. We demonstrate the benefits of this approach in the analysis of single-cell signaling by monitoring caspase activity in individual cells during apoptosis.

JAK protein tyrosine kinases: their role in cytokine signalling

Protein tyrosine kinases (PTKs) are integral components of the cellular machinery that mediates the transduction and/or processing of many extra- and intracellular signals. Members of the JAK family of intracellular PTKs (JAK1, JAK2 and TYK2) are characterized by the possession of a PTK-related domain and five additional homology domains, in addition to a classical PTK domain. An important breakthrough in the understanding of JAK kinases function(s) has come from the recent observations that many cytokine receptors compensate for their lack of a PTK domain by utilizing members of the JAK family for signal transduction.

Biomolecular interaction analysis of IFNγ-induced signaling events in whole-cell lysates: Prevalence of latent STAT1 in high-molecular weight complexes

The basic framework for the JAK/STAT pathway is well documented. Recruitment of latent cytoplasmic STAT transcription factors to tyrosine phosphorylated docking sites on cytokine receptors and their JAK-mediated phosphorylation instigates their translocation to the nucleus and their ability to bind DNA. The biochemical processes underlying recruitment and activation of this pathway have commonly been studied in reconstituted in vitro systems using previously defined recombinant signaling components. We have dissected the Interferon gamma (IFN gamma) signal transduction pathway in crude extracts from wild-type and STAT1-negative mutant cell lines by real-time BIAcore analysis, size-exclusion (SE) chromatography and immuno-detection. The data indicate that in detergent-free cell extracts: (1) the phospho-tyrosine (Y440P)-containing peptide motif of the IFN gamma-receptor alpha-chain interacts directly with STAT1, or STAT1 complexes, and no other protein; (2) non-activated STAT1 is present in a higher molecular weight complex(es) and, at least for IFN gamma-primed cells, is available for recruitment to the activated IFN gamma-receptor from only a subset of such complexes; (3) activated STAT1 is released from the receptor as a monomer.

Intermolecular interactions of the p85alpha regulatory subunit of phosphatidylinositol 3-kinase

The regulatory subunit of phosphatidylinositol 3-kinase, p85, contains a number of well defined domains involved in protein-protein interactions, including an SH3 domain and two SH2 domains. In order to investigate in detail the nature of the interactions of these domains with each other and with other binding partners, a series of deletion and point mutants was constructed, and their binding characteristics and apparent molecular masses under native conditions were analyzed. The SH3 domain and the first proline-rich motif bound each other, and variants of p85 containing the SH3 and BH domains and the first proline-rich motif were dimeric. Analysis of the apparent molecular mass of the deletion mutants indicated that each of these domains contributed residues to the dimerization interface, and competition experiments revealed that there were intermolecular SH3 domain-proline-rich motif interactions and BH-BH domain interactions mediating dimerization of p85α bothin vitro and in vivo. Binding of SH2 domain ligands did not affect the dimeric state of p85α. Recently, roles for the p85 subunit have been postulated that do not involve the catalytic subunit, and if p85 exists on its own we propose that it would be dimeric.

A prominent natural H-2 Kd ligand is derived from protein tyrosine kinase JAK1

The first natural MHC ligand to be sequenced directly was the nonapeptide SYFPEITHI eluted from H-2 Kd molecules of a mouse tumour line, P815 [1]. A GenBank search indicated high homology to a nonapeptide contained within the human tyrosine kinase JAK1: SFFPEITHI, residues 355-363 [2]. This high homology prompted us to look at whether the mouse JAK1 protein has a Tyr residue at position 356 instead of Phe as in the human sequence. Cloning and sequencing of the mouse homologue gene confirmed that this is indeed the case. Thus, the physiological MHC ligand SYFPEITHI is derived from the protein tyrosine kinase, JAK1. The mouse tumor line P815 expresses the 5.4-kb JAK1 mRNA, and the 130,000 kDa JAK1 protein can be readily detected.

Intracellular Signal Transduction: The JAK-STAT Pathway

This book is the first one written about the JAK/STAT pathway. The JAK (Janus Kinase) Protein tyrosine kinases are novel phosphotransferases absolutely required for cellular signalling downstream of non-catalytic cytokine receptors (amongst others). These molecules are components in pathways utilising the STAT (Signal Transducers and Activators of Transcription) transcription factors. The basic components of the JAK/STAT pathway are covered in detail, and the centre piece of the book is a guided tour of the pathway itself. An interesting addition to the book is the chapter on the use of Drosophila melanogaster as a genetic system to probe the pathway at the whole organism level. The book is targeted to researchers who have an interest in intracellular signalling.

Imaging protein interactions by FRET microscopy: FRET measurements by acceptor photobleaching.

This protocol describes the detection of fluorescence resonance energy transfer (FRET) by measuring the quenching of donor emission alone. As opposed to sensitized emission measurements, photobleaching can be performed with high selectivity of the acceptor because absorption spectra are steep at their red edge, allowing the acceptor to be bleached without excitation of the donor. When using acceptor photobleaching FRET measurements, care should be taken that the photochemical product of the bleached acceptor does not have residual absorption at the donor emission and, more importantly, that it does not fluoresce in the donor spectral region. Because of mass movement of protein during the extended time required for photobleaching (typically 1-20 min), it is preferable to perform this type of FRET determination on fixed cell samples. Live-cell FRET measurements based only on donor fluorescence are more feasible using fluorescence lifetime imaging (FLIM), because lifetimes are independent of probe concentration and light path length. The former is not easy to determine in cells, and the latter means that cell shape is not a factor.

Imaging protein interactions by FRET microscopy: FLIM measurements.

This image acquisition protocol is a basic plan for taking a fluorescence lifetime imaging (FLIM) series. FLIM makes live-cell FRET measurements based only on donor fluorescence more feasible, because lifetimes are independent of probe concentration and light path length. The former is not easy to determine in cells, and the latter means that cell shape is not a factor.

Imaging Protein Interactions by FRET Microscopy: FRET Measurements by Sensitized Emission

This protocol describes a method for measuring fluorescence resonance energy transfer (FRET) by the detection of acceptor-sensitized emission. This approach is useful in situations where donor intensities are low and/or there is contamination with high background (auto) fluorescence in the donor channel. However, absorption spectra characteristically exhibit long tails in the higher-energy, shorter-wavelength (blue) region, which may result in the direct excitation of the acceptor molecule in addition to that of the donor, thus resulting in mixing of direct and sensitized emission. Conversely, fluorescence emission tends to tail into the red part of the spectrum, causing donor fluorescence bleed-through into the acceptor detection channel. Corrections for these effects involve the acquisition of fluorescence images of samples containing the donor, the acceptor, and both of these for three different filter settings. The result is an estimation of the sensitized emission, i.e., the emission induced by FRET from the donor to the acceptor alone. Excitation of a donor fluorophore in a FRET pair leads to quenching of the donor fluorescence and increased emission from the acceptor (sensitized emission). This can be normalized using the acceptor emission, measured after specific excitation of the acceptor, to define apparent energy transfer efficiency in each pixel of the image. It is also proportional to the fraction of acceptor molecules that is bound to a donor-tagged molecule. Alternatively, an apparent energy transfer efficiency can also be defined that is proportional to the bound fraction of donor-tagged molecules.