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The Journal of Immunology, 2003, 171: 519-523.
Copyright © 2003 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: A Chemical Genetic System for the Analysis of Kinases Regulating T Cell Development 1

Angela Denzel*, Katherine J. Hare{dagger}, Chao Zhang{ddagger}, Kevan Shokat{ddagger}, Eric J. Jenkinson{dagger}, Graham Anderson2,{dagger} and Adrian Hayday2,3,*

* Department of Immunobiology, New Guy’s House, Guy’s, King’s and St. Thomas’s School of Medicine, London, United Kingdom; {dagger} Department Anatomy, Medical Research Council Center for Immune Regulation University of Birmingham, Birmingham, United Kingdom; and {ddagger} Department of Cellular and Molecular Pharmacology, University of California San Francisco, CA 94143


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
To understand the regulatory activities of kinases in vivo requires their study across a biologically relevant window of activity. To this end, ATP analog-sensitive kinase alleles (ASKAs) specifically sensitive to a competitive inhibitor have been developed. This article tests whether ASKA technology can be applied to complex immunological systems, such as lymphoid development. The results show that when applied to reaggregate thymic organ culture, novel p56Lck ASKAs readily expose a dose-dependent correlation of thymocyte development with a range of p56Lck activity. By regulating kinase activity, rather than amounts of RNA or protein, ASKA technology offers a general means for assessing the quantitative contributions to immunology of numerous kinases emerging from genomics analyses. It can obviate the generation of multiple lines of mice expressing different levels of kinase transgenes and should permit specific biological effects to be associated with defined biochemical activities.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The pivotal role of kinases in biology is clear (1, 2). Nonetheless, we are often ignorant of their biologically relevant substrates and the quantitative window of kinase activity required to phosphorylate them. It is seldom clear when biological processes have graded responses to kinase dosage or "all-or-nothing" responses to thresholds of kinase activity, and thus it is difficult to predict the effect of modest differences in kinase activity that will characterize human genetic variation.

An improved understanding ideally requires a capacity to regulate specific kinase activities within complex mammalian systems. In this regard, a recent technological innovation described ATP analog-sensitive kinase alleles (ASKAs)4 that can accommodate ATP and function normally, but that additionally have high affinity for large ATP analogs, such as 4-amino-1-tert-butyl-3-(1'-naphthyl)pyrazolo[3,4-d]pyrimidine (NaPP1), that compete for entry into the ATP binding site (3, 4). By contrast, the active site of natural kinases is too small to accommodate such inhibitors (3) (Fig. 1a). Thus, ASKA technology theoretically permits one to regulate the biological effects of any chosen kinase.

This notwithstanding, the application of ASKA technology to complex mammalian biology faces potentially serious uncertainties. These include the expression levels of ASKAs achievable in primary cells and whether the doses of inhibitor needed to gain access to relevant cells within a multicellular organ cause nonspecific biological dysregulation: in sum, will the achievable expression levels of the ASKAs and the practical concentrations of inhibitor combine to regulate kinase activity across a biologically relevant range? To resolve these issues, this article examines the practicality of ASKA technology for studying T cell development in reaggregate fetal thymic organ culture (RTOC).

Stages of thymocyte development are readily marked by CD4 and CD8 expression (5). Early CD4-CD8- "double-negative" (DN) thymocytes mature into CD8low cells (immature single-positive cells (ISPs)) that become CD4CD8 double-positive (DP) (6), and finally CD4+ or CD8+ single-positive (SP) cells. The DN-ISP-DP transition depends on the TCR {beta}-chain contributing to a pre-TCR (7, 8, 9), while the DP-SP transition depends on TCR{beta} contributing to a mature {alpha}{beta} TCR. The pre-TCR and TCR{alpha}{beta} are each associated with the src-related kinases, Lck and Fyn (10, 11, 12).

Several lines of transgenic and gene knockout mice have indicated that the pre-TCR-dependent DN-ISP-DP transition is inhibited by Lck overexpression as well as by Lck deficiency or expression of a dominant-negative Lck (13, 14, 15). Nonetheless, conclusions are inevitably complicated by variability in the onset and durability of Lck transgene expression in the different mouse strains and by the uncertain relationship between Lck expression and Lck activity. Instead, it would be useful to show that different biological effects occur within a single system as Lck activity is incrementally altered across a continuous range. This seemed an ideal context in which to test the application of ASKA technology to a complex immunological process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Retroviral infections

Lck alleles generated using QuikChange Site-directed mutagenesis (Stratagene, La Jolla, CA) were subcloned into the pMX vector (16). Briefly, 2 x 106 phoenix-E packaging cells were transfected with 20 µg of vector DNA in CaCl2 and 2x HBS (pH 7.05). Medium was changed 8 h after infection, and supernatant viral particles were harvested at 48, 72, and 96 h, passed through 45-µm filters; aliquoted, and stored at -70°C (17).

RTOC, transduction, and formation

Embryonic thymocytes and stromal cells were isolated and prepared as described previously (18). E14 thymocytes were centrifuged at 2100 rpm for 1 h, incubated with virus for 3 h, and pelleted by centrifugation with thymic epithelial cells at a ratio of 1:3. The resultant slurry was transferred by a finely drawn glass pipette to the surface of a 0.8-µm nucleopore filter (19). Cultures were harvested at indicated times and thymocytes were released from reaggregates using fine cataract knives.

Transduction and Western blot of thymoma BW5147

Briefly, 1.2 x 106 cells were transduced with control vector or Lcka-as and, after 48 h, gfp+ cells were flow cytometry sorted and expanded in culture with 0, 0.1, 1, 5, or 10 µM NaPP1 (Cellular Genomics, Branford, CT) for another 48 h before lysis in Nonidet P-40 buffer. One hundred micrograms of protein lysate was separated on 10% SDS gels, blotted onto nylon membranes, and probed using Abs against total Lck (Santa Cruz Biotechnology, Santa Cruz, CA) and phosphorylated Ysrc416 (Cell Signaling, Beverly, MA), respectively.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Lcka-as is an active yet inhibitable allele of Lck

Mutation to glycine of Thr316 in the ATP binding site of Lck creates Lck-as (Fig. 1a), an allele predicted to bind ATP and function as a normal tyrosine kinase (20, 21), but that should also accommodate NaPP1, an enlarged analog of the Src family kinase inhibitor PP1 that competes with ATP for entry into the ATP binding site (3). Other naturally occurring kinases contain a large amino acid corresponding to T316 that precludes NaPP1. Following construction of Lck-as, we combined the T316 G mutation in cis with a Y505 F mutation (Lcka) that renders Lck constitutively active (22), to create Lcka-as



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  		FIGURE 1. a) Schematic representation of inhibition by NaPP1. T316 was exchanged for glycine, creating a unique hole in the ATP-binding pocket of Lck. ATP can normally access the ATP binding site and Lck can phosphorylate downstream substrates. However, in the presence of NaPP1, there is competition for the ATP binding site of the ASKA (Lck-as), blocking downstream substrate activation. Nonmutated kinases preclude access to NaPP1. b and c, BW 5147 thymoma cells were transduced with Lcka-as and gfp+ cells were incubated with either 0, 0.1, 1, 5, or 10 µM NaPP1 for 48 h. Western blots were probed with Abs to detect Lck (b) and Y394-phosphorylated Lck (c).

 
A primary activity of Lck is autophosphorylation. To test Lcka-as activity, several T cell lines were transduced with retroviruses expressing Lcka-as from a polycistronic RNA also encoding gfp from an internal ribosome entry site. Following transduction, cells were incubated for 48 h in different NaPP1 concentrations, after which equal amounts of gfp+ cell lysates were Western blotted and probed with Abs for either total p56Lck (Fig. 1b) or Y394-phosphorylated Lck (Fig. 1c). As the inhibitor was increased, total Lck was comparably expressed (serving as a control for protein loading), whereas phosphorylated p56Lck decreased in amount in a dose-dependent fashion. Thus, Lcka-as encodes a novel form of Lck, the activity of which is regulatable with NaPP1.

Lcka-as is biologically active and specifically inhibitable in wild-type (wt) RTOC

DN thymocytes were transduced with a virus expressing gfp only, or coexpressing Lcka, and Lcka-as, respectively, and incubated in RTOC for ~5 days, by which time >50% of gfp+ thymocytes transduced with a gfp-only virus developed to the DP stage (Fig. 2a; in all plots, the x-axis depicts CD8 expression, the y-axis depicts CD4). Although DP development varied from 50% to 82% in different experiments (e.g., because of age variation in the fetuses used as sources of thymocytes and stroma, or variable time of harvest), results within single experiments were more consistent. Moreover, development was largely comparable in nontransduced cultures, "gfp-only" transduced cultures and nontransduced (gfp-) cells recovered from RTOC supporting gfp+ transduced cells (Fig. 2). Thus, neither transduction nor gfp expression substantially affects thymocyte development in RTOC.



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  		FIGURE 2. a, RTOC from wt mice transduced with control (gfp-only) vector, Lcka, or Lcka-as, harvested after 5 days with or without 10 µM NaPP1, counted (control: 4.5 x 105 cells; Lcka no NaPP1: 4.2 x 105; Lcka 10 µM NaPP1: 4.4 x 105; Lcka-as no NaPP1: 4.1 x 105; Lcka-as 10 µM NaPP1: 4.2 x 105), and assayed for CD4 (y-axis) and CD8 (x-axis). Numbers in upper right quadrants show the percentage of thymocytes expressing CD4 and CD8; numbers in parentheses show the percentage of CD3low cells. b, CD4 and CD8 analysis of RTOC from wt mice transduced with control vector or with Lcka-as, harvested after 9 days with or without 10 µM NaPP1. Numbers in the upper right quadrants show the percentage of DP thymocytes; numbers in lower right quadrants show the percentage of ISPs. c, CD4 and CD8 expression on thymocytes from wt mice transduced with Lcka-as and cultured for 5 days in RTOC with indicated concentrations of NaPP1. Top panels, Analyses of gfp+ cells and lower panels, analyses of gfp- cells from the same RTOCs. (cell yields - control: 2.2 x 105; Lcka-as no NaPP1: 2.4 x 105; Lcka-as 0.5 µM NaPP1: 1.9 x 105; Lcka-as 2 µM NaPP1: 3.2 x 105; Lcka-as 10 µM NaPP1: 2.4 x 105; control: 1.7 x 105 cells). d, Percentage of DP cells among gfplow vs gfphigh thymocytes at different inhibitor concentrations. The left-most points show the range of DP development (78–82%) in gfp-only transduced cultures in this experiment.

 
Consistent with the deleterious effects of Lck overexpression in transgenic mice (15), gfp+ thymocytes transduced with an activated Y505 F Lck allele (Lcka) showed almost complete inhibition of the DN-DP transition (4% DP), as did Lcka-as-transduced thymocytes (Fig. 2a), thereby demonstrating the ASKA to be biologically equivalent to Lcka. However, the biological activity of Lcka-as was substantially inhibited by addition to the RTOC of 10 µM NaPP1. Thus, the inhibitor rescued the generation of DPs (40%) and the expression by many of them of low levels of CD3 (>23% compared with 18% in controls; Fig. 2a). The inhibitor’s specificity was evident in its failure to rescue RTOCs expressing Lcka (Fig. 2a). RTOCs harvested at 9 days also showed strong inhibition of DP maturation by Lcka-as as well as an accumulation of CD8+ ISPs (48%), previously associated with Lck overexpression (15). Both effects were substantially reversed by 10 µM NaPP1 (Fig. 2b). In sum, the combined use of a novel Lck ASKA and an ATP analog inhibitor provided a simple means to alter the activity of Lck over a biologically relevant range within a complex organ culture.

The inhibitory effect of activated Lck is poorly understood, but it is not readily attributed to apoptosis since transgenic mice expressing increased levels of wt or activated Lck showed no obvious decrease in thymocyte numbers (15). Similarly, comparable numbers of gfp+ thymocytes were recovered from the gfp-only and from the Lcka-as-transduced cultures. The recovery of similar numbers of cells from cultures containing 10 µM NaPP1 indicated that inhibitor per se also does not affect cell viability (see Fig. 2a).

To determine whether T cell development shows a graded or an all-or-nothing threshold response to variation in Lck activity, different doses of inhibitor were added to RTOCs. In this experiment, the representation of DPs in control vector-transduced cultures ranged from 53 to 71%, whereas for Lcka-as-transduced cells it was ~9% (Fig. 2c). Increasing NaPP1 gradually reduced Lck overactivity, incrementally restoring DP cells to 11% (0.5 µM NaPP1), 20% (2 µM), and ~45% (10 µM) (Fig. 2c). The development of gfp- cells from the same RTOC was within the range of the controls (Fig. 2c) and comparable cell yields again suggested that cell viability was not greatly affected by Lcka-as or by 10 µM inhibitor (see legends to Figs. 2 and 3). Thus, the dose-dependent regulation of a Lck ASKA in RTOC readily exposed a quantitative correlation of T cell development with Lck activity across a continuous range.



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  		FIGURE 3. a, Development of RAG1-/- thymocytes assessed by flow cytometry for CD4 (y-axis) and CD8 (x-axis) following transduction with wt Lck, Lcka or Lcka-as. b, CD4 and CD8 expression on Lcka-as-transduced thymocytes from RAG1-/- mice cultured in indicated concentrations of NaPP1. Number of cells harvested from individual RTOCs: control: 1.5 x 105 cells; Lcka-as no NaPP1: 1.9 x 105; Lcka-as 2 µM NaPP1: 1.8 x 105; Lcka-as 5 µM NaPP1: 1.9 x 105; Lcka-as 10 µM NaPP1: 1.8 x 105.

 
Fig. 2d plots inhibitor concentrations against DP development for Lcka-as-transduced thymocytes in another RTOC in which cells expressing high or low gfp levels were analyzed separately. Since the Lck and gfp cistrons are linked, most gfplow cells express low Lck levels, while gfphigh cells generally express more Lck. Consistent with this, comparable DP development (~42%) is seen in gfplow cultures with no inhibitor and in gfphigh cultures with 5 µM inhibitor. However, the enhanced thymocyte maturation, even of gfplow cells, at high inhibitor concentrations (~70% DPs) demonstrates that ASKA methodology can reduce Lck biological activity to below that achieved by gating for gfplow expression. In sum, the application of ASKA technology permits Lck activity to be regulated over an unprecedentedly broad, biologically relevant range in a complex organ culture.

Lcka-as is biologically active and specifically inhibitable in mutant RTOC

The biological function of Lcka-as was independently assayed in RTOC using thymocytes from RAG1-deficient mice that cannot rearrange TCR{beta} genes and therefore cannot undergo DN-ISP-DP maturation (23). Thus, gfp-only vector-transduced thymocytes remain >90% DN (Fig. 3a), whereas enforced Lck expression mimics pre-TCR signaling, promoting the appearance of ISPs and increasing DP representation from 0.2 to 16% (Fig. 3a). Although transgenic Lck was reported to rescue DP development more fully than this (24), those studies used a debilitated Lck. Instead, Lck overexpression in transgenic mice led primarily to ISP accumulation (15). This distortion in thymocyte development was exaggerated in Lcka and Lcka-as-transduced RTOCs in which DP rescue was only 5–7% (Fig. 3a). However, increasing doses of inhibitor achieved an incremental rescue of Lcka-as-transduced RAG1-/- DPs (13, 31, 37, 43%; Fig. 3b). Thus, ASKA technology permitted the dose-dependent regulation of thymocyte development by Lck in wt and mutant forms of RTOC.

Chemical genetic complementation of Lck deficiency across a complete range

Finally, the Lck-ASKA was examined for its capacity to complement the defective development of Lck-/- thymocytes. Lck-/- mice show partial inhibition of the DN-ISP-DP transition (~35% DP cells) because the defect in pre-TCR signaling is partly compensated by fyn (Fig. 4a and Ref13). Rather than rescue this deficiency, strong Lck overexpression permitted only 1% DPs, consistent with its inhibition of thymocyte development (Fig. 4d). However, by selecting for lower gfp expression (lower levels of Lck), DP representation went from 7% (Fig. 4c) to 27% (Fig. 4b) and to 47% (Fig. 4a). However, substantial rescue of DPs (46%) was also achievable among gfphigh cells, simply by adding 10 µM NaPP1 (Fig. 4d). Indeed, within a single NaPP1-treated RTOC, the complementation of Lck deficiency was achieved across a full biologic range. Thus, cells gated for "medium" gfp in the presence of inhibitor showed essentially complete rescue (~60% DP; Fig. 4, b and c), whereas gfplow cells defined thymocytes in which Lck activity was now insufficient, and in which DP representation fell back to 38% (Fig. 4a).



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  		FIGURE 4. CD4 (y-axis) and CD8 (x-axis) expression on Lcka-as-transduced Lck-/- thymocytes in RTOC cultured with or without 10 µM NaPP1 inhibitor. In each section (a–d), the top panel shows gating on gfp expression (a, low; b, low-medium; c, medium-high; d, high); the middle panel shows CD4 and CD8 analysis; and the bottom panel shows forward scatter fluorescence for gated ISPs (left panels) or DPs (right panels). The mean fluorescence intensity (MFI) for each analysis is noted. An analysis of gfp- cells from the same culture is shown in the center of the top row.

 
Excess Lck favors cell activation over differentiation

Although overexpressed Lck inhibits TCR{beta} chain gene rearrangement (25), the inhibition of T cell development in Lck-overexpressing mice does not phenocopy RAG deficiency and its underlying nature has not been fully clarified. There is little evidence for excess apoptosis (see above), and it has been noted in transgenic studies and here that there is an associated increase in ISPs (15). The higher the Lck expression (gfphigh and 0 inhibitor; Fig. 4d), the greater was ISP accumulation (60%), whereas in the presence of inhibitor, DP representation exceeded ISP representation in all but the gfplow cells (Fig. 4). Analysis of forward scatter (Fig. 4) and other properties showed that the ISPs are activated blast cells, whereas DPs are mostly smaller and resting. Of note, as Lck activity was gradually increased, the ISPs were more activated, as reflected by increasing forward scatter (600–750; Fig. 4). Therefore, increasing Lck activity in early thymocytes appears, at least in part, to skew pre-TCR signaling toward cell activation at the expense of differentiation. This seems consistent with the role of activated Lck in malignancy (22). The capacity to sort and analyze cells expressing different levels of Lck from ASKA-transduced cells cultured in different inhibitor concentrations now offers the potential to identify protein phosphorylation and/or gene expression patterns that correlate with particular levels of kinase activity and their associated biological outcomes.

In sum, we have combined a novel chemical genetic approach with retroviral transduction of RTOC to achieve rapid and efficient analysis of a kinase playing a crucial role in mammalian cells. Previous study of the dose-dependent effects of Lck on T cell development required the labor-intensive generation of multiple lines of transgenic mice expressing different levels of an Lck transgene (15). Although those studies provided a firm foundation to validate the use of ASKA as a tool in developmental immunology, it is already clear from Fig. 2d that the range of biological effects of Lck achievable with ASKA technology exceeds that obtained by selecting cells stochastically expressing different levels of a kinase. Indeed, whereas identification of cell lines or transgenic mice expressing a biologically relevant level of a transgene can be problematic, the capacity to use ASKAs to regulate kinase activity may allow one to use mice or cell lines grossly overexpressing a particular kinase. The relative ease of ASKA construction (successfully applied to v-Src, CDK2, Fus3, CAMKII{alpha} (4)), the specificity of the inhibitor’s effects; the broad range of kinase activity achievable, and the closer correlation of biological outcome with kinase activity, as opposed to inducible levels of gene expression (26, 27) suggest that ASKA technology should be usefully applied to many kinases emerging from the expression profiling of lymphoid cells (28, 29) and from genome sequencing.


   Acknowledgments
 
We thank Suzanne Creighton, Wayne Turnbull, and Drs. M. J. Bijlmakers, D. Pennington, and E. Hoffman.


   Footnotes
 
1 This work was supported by the Wellcome Trust (to A.H. and A.D.), the Medical Research Council (to E.J.J. and G.A.), and the National Institutes of Health (to K.S.). Back

2 G.A. and A.H. contributed equally to this study. Back

3 Address correspondence and reprint requests to Dr. Adrian Hayday, Department of Immunobiology, New Guy’s House, Guy’s King’s, and St. Thomas’s School of Medicine, London SE1 9RT, United Kingdom. E-mail address: adrian.hayday{at}kcl.ac.uk Back

4 Abbreviations used in this paper: ASKA, analog-sensitive kinase allele; NaPP, 4-amino-1-tert-butyl-3-(1'-naphthyl)pyrazolo[3,4-d]pyrimidine; RTOC, reaggregate fetal thymic organ culture; DN, double negative; ISP, immature single positive; DP, double positive; SP, single positive; wt, wild type. Back

Received for publication January 15, 2003. Accepted for publication May 13, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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A. Giannini and M.-J. Bijlmakers
Regulation of the Src Family Kinase Lck by Hsp90 and Ubiquitination
Mol. Cell. Biol., July 1, 2004; 24(13): 5667 - 5676.
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