The orphan adapter protein SLY1 as a novel anti-apoptotic protein required for thymocyte development
© Reis et al; licensee BioMed Central Ltd. 2009
Received: 21 February 2009
Accepted: 15 July 2009
Published: 15 July 2009
SH3 containing Lymphocyte Protein (SLY1) is a putative adapter protein exclusively expressed in lymphocytes which is involved in antigen receptor induced activation. We previously have generated SLY1Δ/Δ mice harbouring a partial deletion in the N-terminal region of SLY1 which revealed profound immunological defects in T and B cell functions.
In this study, T cell development in SLY1-/- and SLY1Δ/Δ mice was analysed ex vivo and upon cultivation with the bone marrow stromal cell line OP9. SLY1-deficient thymocytes were compromised in inducing nutrient receptor expression and ribosomal protein S6 phosphorylation, indicating a defect in mTOR complex activation. Furthermore, SLY1 was identified as a novel anti-apoptotic protein required for developmental progression of T cell precursors to the CD4+CD8+ double-positive stage by protecting from premature programmed cell death initiation in developing CD4-CD8- double-negative thymocytes. In addition, SLY1 phosphorylation was differentially regulated upon Notch ligand-mediated stimulation and expression of the preTCR.
Thus, our results suggest a non-redundant role for SLY1 in integrating signals from both receptors in early T cell progenitors in the thymus.
T cell development mainly takes place in the thymus and is characterized by defined developmental stages ultimately leading to the generation of mature αβ and γδ T cells. The earliest T cell progenitors entering the thymus via the bloodstream lack CD4 and CD8 expression and are therefore called double-negative (DN). DN thymocytes can be further subdivided into four successive developmental stages according to the expression of CD25 (IL-2 receptor α chain) and CD44 (Pgp-1) [1, 2]. The most immature progenitors (DN1) are assigned to the CD44+CD25- subset. Upregulation of CD25 marks progression to the DN2 stage (CD44+CD25+). Irreversible commitment to the T cell lineage is not established until the DN3 stage, which can be identified as the CD44-CD25+ DN subset. In this cell population, rearrangement of the TCRβ- or TCRγδ genes is accomplished, leading to either development into the αβ- or the γδ lineage. For αβ T cells, a successfully generated TCRβ chain leads to formation of the preTCR via pairing with the preTα chain and CD3 signalling complexes. Thymocytes failing to generate a preTCR undergo programmed cell death . Thymocytes otherwise destined to induce programmed cell death are rescued from apoptosis upon preTCR generation. This event is termed β-selection, leading to extensive cellular expansion and cessation of further TCRβ locus recombination (allelic exclusion). Cells which have passed this developmental checkpoint downregulate CD25 to become DN4 cells and then proceed to the numerically dominant CD4and CD8 double-positive (DP) stage. Lymphocytes evading cell death are frequently the origin of malignant transformations [4, 5]. Although of high clinical relevance, the regulation of pro- and anti-apoptotic processes at this specific stage of development is still incompletely understood.
In the past years, it has been established that for commitment to the T cell lineage Notch signalling is essential [6–9]. In combination with the preTCR, Notch induces proliferation and differentiation to DP thymocytes [10, 11]. More recently, Notch ligand engagement was additionally shown to be crucial for controlling progenitor cell metabolism by activating Phosphatidyl-Inositol-Kinase-1 (PDK-1), Akt and the mammalian Target of Rapamycin (mTOR) complex [12, 13]. Notch- and preTCR-dependent upregulation of mTOR induces expression of the nutrient receptors CD71 (transferrin receptor) and CD98 (amino acid transporter). This is, together with the PDK-1- and mTOR-dependent activation of AGC-serine kinases like S6 Kinase 1 and Ribosomal S6 Kinase, a key event for cell mass increase and induction of a proliferation-competent status, eventually leading to six to ten consecutive rounds of cell division after passage of the β-selection checkpoint.
SH3 Lymphocyte Protein (SLY1) is a putative adapter protein containing a SH3- and SAM-domain. It constitutes the first described member of a family of highly homologous proteins conserved in mammals whose molecular function is still elusive. The protein SLY1 consists of 380 amino acids and is exclusively expressed in lymphocytes . It has been originally found in an adhesion assay screen using a T cell lymphoma cDNA library. SLY1 has been independently identified by another group during a screen for new serine kinase substrates . There it was shown that SLY1 is specifically phosphorylated upon T cell receptor (TCR)-triggering or stimulation with phorbol esters at Serin27. This phosphorylation could be prevented by Protein Kinase C (PKC)- or PI3Kinase-inhibitors, however, the directly phosphorylating kinase has not been identified to date. Further hints for a possible involvement of SLY1 in the TCR-transduction pathway were delivered by the immune-compromised phenotype of mice expressing a truncated SLY1 protein. This truncation resulted in aberrant subcellular localisation of the remaining protein SLY1Δ which is lacking part of the bipartite nuclar localisation sequence and the known phosphorylation site . Furthermore, it was shown that SLY1 plays a substantial role in the development and activation of immune cells, although the underlying mechanism of this phenotype could not be clarified [16, 17].
Two additional highly homologous proteins have been described: SLY2, also termed SAMSN1/NASH1/HACS1, is more broadly expressed in haematopoietic tissues, endothelial cells and myeloid leukemias and myelomas [18, 19]. SLY2 has been implicated in inducing a plasma-cell-like phenotype when overexpressed in B cells and has been linked to the inhibitory receptor PirB . The SLy2 locus lies in a region which is frequently disrupted in translocation events leading to haematopoietic malignancies . Most recent data suggest the involvement of SLY2 as tumour suppressor in human lung cancer . SLY3 (SASH1) is expressed in almost all tissues and has been repeatedly described as a tumour suppressor based on its frequent downregulation in breast cancer and colon carcinoma [22, 23].
In this study, mice harbouring a complete deletion of SLY1 protein were generated and thymocyte development was subsequently analysed. Thereby, an important role for the orphan adapter protein SLY1 at the thymocyte DN to DP progression was identified, leading to a reduction in thymic cellularity by about 50%. SLY1-deficient thymocytes were compromised in inducing nutrient receptor expression and ribosomal protein S6 phosphorylation, indicating a defect in mTOR complex activation and suggesting a role for SLY1 in integrating signals derived from the preTCR and from the Notch receptor. This defect resulted in abnormally increased deletion of precursor cells by induction of programmed cell death, yielding a strong reduction in DP thymocyte numbers. Interestingly, SLY1Δ mice expressing a protein harbouring a deletion of 81 amino acids in the N-terminal region of SLY1 exhibited an identical phenotype. Thus, a non redundant role for this region comprising the phosphorylation site and part of the nuclear localisation sequence (NLS) could be assigned.
Reduced cellularity of lymphoid organs
Delayed T cell development
Defect in proliferation and differentiation in vitro
Partially impaired activation of mTOR-complex in SLY1-defective DN thymocytes
Increased apoptosis induction in SLY1-defective DN3 thymocytes
Regulation of phosphorylation of SLY1 in thymocytes
T cell development is a tightly regulated process involving numerous successive steps of differentiation. Of particular importance for the maturation of αβ T cells is the successful rearrangement of the VDJ genes coding for the TCRβ chain which then pairs with the preTα chain to initiate preTCR signalling [1, 27–29]. In the present work, induction of programmed cell death in DN3 thymocytes could be precisely recapitulated as being principally dependent on intracellular preTCR expression, thereby supporting preTCR-signalling as the major decisive constraint in death versus survival decision. Furthermore, the orphan adapter protein SLY1 was identified as a novel anti-apoptotic protein required for developmental progression of T cell precursors to the DP stage. This anti-apoptotic function resulted in protection of thymocytes from premature programmed cell death initiation at the DN3 thymocyte stage. As a consequence, thymocyte DP number and therefore total thymus cellularity was severely reduced in SLY1-targeted mice.
Haematopoietic progenitors entering the thymus via the bloodstream depend on a sustained Notch receptor-ligand engagement to develop towards the T cell lineage [2, 6, 30, 31]. The OP9 differentiation system can be used to recapitulate the signals required for haematopoietic precursor cells to undergo T cell development until the DP thymocyte stage in vitro. This differentiation system was used to further elucidate the underlying defect responsible for the reduced cellularity of SLY1-/- and SLY1Δ/Δ thymi. As anticipated from the ex vivo situation, a reduction in proliferation and differentiation upon OP9 culture relative to littermate controls was found. Thus, a migration defect of progenitor cells with a subsequent reduction in thymic repopulation capacity as the major cause for the resulting defect in thymic cellularity could be excluded. Furthermore, it could be clearly shown that the reduction in thymocyte proliferation is cell intrinsic and that the principal defect is not influenced by differences in thymic environment derived from stromal cells.
Yet Notch signalling is not only mandatory for commitment to the T cell lineage. Recently, it was also shown to be crucial for inducing a replication-competent status in thymocytes once preTCR signalling is initiated [10, 12, 13]. A prerequisite for cellular expansion is an increased metabolic activity including elevated expression of nutrient receptors and protein synthesis. The activity of mTOR has a key role in controlling this process. In peripheral T cells, growth factors have been reported to modulate Akt and mTOR activity and thereby to increase survival of T cells . Growth factor withdrawal resulted in metabolic collapse and loss of nutrient receptor expression. In SLY1-/- and SLY1Δ/Δ thymocytes, induced expression of the nutrient receptors CD71 and CD98 as well as S6 Kinase activation were impaired relative to wt littermates. This diminished mTOR activity correlated with a reduced cell size increase. Therefore a defect in metabolic activity in SLY1-defective thymocytes can be inferred, leading to increased cell death rate and concomitant reduction of thymic cellularity.
Full mTOR activation in DN thymocytes is dependent on both Notch- and preTCR-derived signals [10, 12, 13]. There is limited insight into the signal integration process of both pathways in thymocytes. Current literature data suggests that signal integration occurs at the level of PI3Kinase activation. Notch signalling is thought to decrease expression of PTEN, the negative regulator of PI3Kinase activation . preTCR-formation is thought to activate Akt, probably also via PI3Kinase activation . It is not precisely clear how the presence of a preTCR without ligand engagement leads to the activation of Akt, and whether there are also more direct interactions of the Notch pathway with the PI3Kinase pathway which are not regulated by transcription. The present work may add insight into how the different signals are processed by connecting them with the orphan adapter protein SLY1. The reduced frequency of SLY1-/- and SLY1Δ/Δ progenitor cells with increased metabolic activity suggests a role for SLY1 in transmitting signals from either the preTCR or the Notch receptor to mTOR, or even for integrating signals from both receptors. As opposed to reports from peripheral T lymphocytes showing that SLY1 phosphorylation is strictly dependent on TCR-stimulation, we have shown in this study that SLY1-phosphorylation in thymocytes is independent of the preTCR. Interestingly, we have found that the phosphorylation of SLY1 at serine27 is differentially regulated upon Notch-stimulation dependent on the absence or presence of preTCR-signalling. This differing phosphorylation could be interpreted as a signal integration process of the two converging pathways downstream of the Notch receptor and downstream of the preTCR.
When cultured on OP9 DL-1 and OP9 GFP control cells, the anti-apoptotic function of SLY1 was independent of preTCR- and Notch-derived signals, indicating a general pro-survival role for SLY1 protein independent of both receptors. However, OP9 GFP cells have been reported to express mRNA for the Notch ligands jagged-1 and jagged-2, although in low amounts . Therefore, it cannot be excluded that there might still be residual Notch-signals available to thymocytes cultivated on OP9 GFP control cells. In addition, OP9 cells secrete or present other unknown factors required by haematopoietic precursor cells. These residual signals induced by unknown factors could therefore transmit anti-apoptotic signals which are not properly transmitted in SLY1-defective thymocytes cultivated on OP9 GFP cells compared to the situation in SLY1+/+ thymocytes. In addition, freshly isolated thymocytes have already been receiving Notch signals during their sojourn in the thymus in vivo, and therefore the increased apoptosis induction of SLY1-defective thymocytes on OP9 GFP cells compared to SLY1+/+ thymocytes could already have been initiated in the thymus in vivo before isolation of the cells. Yet unexpected is the observation that Notch signalling can even drive thymocytes into apoptosis if they fail to generate a preTCR as observed upon OP9 culture of DN3 thymocytes in the present work. In contrast to this notice, Notch signalling in DN3 thymocytes is thought to play a general anti-apoptotic role by activating the Akt kinase pathway . This study therefore adds additional complexity to the effector functions of Notch signalling in DN3 thymocytes, implying a pro-apoptotic outcome of Notch receptor activation in a certain cellular context.
As previously described, SLY1Δ/Δ mice expressing a SLY1 protein harbouring a partial deletion in the N-terminal region show impaired immune responses [16, 17]. The region missing in SLYΔ contains part of a bipartite nuclear localisation sequence and the known phosphorylation site closely associated to the potential localisation sequences. As a consequence, the subcellular localisation of SLY1Δ is shifted from a nucleocytoplasmic to a strictly cytoplasmic localisation . The putative protein-protein interacting SH3- and SAM-domains remain intact in SLY1Δ, possibly still enabling its interference with other signalling proteins. However, a dominant negative effect of SLY1Δ can be excluded due to the comparable phenotype of SLY1-/- mice. One conclusion that can be drawn from this data is that an essential function, at least for T cell development, of SLY1 is mediated by the amino acids at position 20–100. An apparent role for this deleted region could be either to regulate the correct subcellular localisation of the protein or to be directly required for interaction with SLY1 binding partners by providing a sequence specific recognition signal.
In the present work, SLY1 is described as a novel anti-apoptotic protein required for thymocyte development by preventing DN thymocytes from premature initiation of programmed cell death. SLY1-negative thymocytes exhibited impaired activation of the mTOR complex, supporting an essential role of mTOR in thymocyte development. We therefore propose a role for SLY1 in signal integration of the Notch receptor and preTCR pathways culminating in the activation of the mTOR protein complex in developing thymocytes. As the sly1 gene is located on the X-chromosome in man, its function could be either negatively affected in some cases of x-linked immunodeficiency or activating mutations could contribute to lymphoma development. In the future, the identification of the interacting kinase and the precise molecular mechanism by which SLY1 contributes to mTOR activation and prevention of programmed cell death will be of great interest.
RNA isolation and RT-PCR
Total RNA from cells was isolated using Trizol Reagent (Invitrogen) according to the manufacture's instructions. First-strand cDNA synthesis was performed using 1 μg of total RNA with M-MLV reverse transcriptase and oligo dT primer (Invitrogen).
SLY1 transcripts were amplified with SLY1_exon6_forward: 5'-CCC GGA GGA TTC TGG GAA GA-3' and SLY1_exon8_reverse: 5'-GAA GTC AGT GTG GAC TCG GG-3'. The following primer sequences were used to amplify GAPDH: (forward) 5'-CAT GTA GGC CAT GAG GTC CAC CAC-3' and (reverse) 5'-TGA AGG TCG GTG TGA ACG GAT TTG GC-3'.
Gene targeting and mice
Generation of SLYΔ/Δ mice and genotyping has been described previously . For generation of SLY-/- mice, E14.1 embryonic stem (ES) cells from 129/Ola mice were grown in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) supplemented with 2 mM glutamine (Seromed, Wien, Austria), leukemia inhibitory factor, 100 U/ml penicillin, 100 μg/ml streptomycin (Seromed), 50 μM 2-mercaptoethanol (Invitrogen) and 15% heat-inactivated fetal bovine serum (Pan Biotech, Aidenbach, Germany). Genomic fragments flanking the murine sly1 gene as well as neomycin and thymidine kinase were cloned into pBluescript (Agilent technologies, Santa Clara, CA, USA) and fully sequenced. The targeting vector was designed in such a way that the neomycin resistance cassette was inserted in reverse orientation in exon 1 of the sly1 gene 3' of the starting ATG without deleting any endogenous sequence. E14.1 ES cells were electroporated with the NotI-linearized targeting vector, and the transfected cells were subsequently subjected to G418 and ganciclovir selection. Clones carrying the correct homologous recombination were identified by Southern blot hybridization with the 5' flanking probe indicated in Figure 1 after digestion of ES cell DNA with EcoRV and BamHI. Single integration was verified by probing the Southern blot with the neomycin resistance cassette. Correctly targeted ES cell clones were injected into C57BL/6 blastocysts, which were transferred into pseudopregnant foster mice. Resulting chimeric mice were backcrossed to C57BL/6 mice, and germ line transmission of the targeted allele was again confirmed by Southern blot analysis. SLY1-/- mice were backcrossed to the C57BL/6 background for six to ten generations. Wild-type littermates were used as controls. Genotyping of SLY1 knockout mice was performed by PCR with the following primers: 5'-TGA CGG CAG TAG GGA TGG TAG-3' (forward); 5'-CGC CTT CTT GAC GAG TTC TTC T-3' (neo reverse); 5'-AGT GGC CTG GGG GAG ATG T-3' (Wt reverse). SLY1Δ/Δ mice were backcrossed for twelve generations.
Mice were kept according to national guidelines for animal care in an SPF animal facility. All animal work was performed according to the guidelines of the German national animal care regulations. RAG1-/- mice were purchased from Jackson Laboratories.
Flow cytometric analysis
Antibodies conjugated to fluorescein isothiocyanate, phycoerythrin, allophycocyanin, percp, phycoerythrin-cy7, allophycocyanin-cy7 and biotin were obtained from either Pharmingen (San Diego, CA, USA), Southern Biotech (Birmingham, Alabama, USA) or eBioscience (San Diego, CA, USA). Alexa-fluor488-conjugated anti-phospho-Akt and anti-phospho-S6 antibodies were obtained from Cell Signalling (Danvers, MA, USA). Cells were stained with saturating concentrations of antibody in accordance with the manufacturer's instructions. Data were acquired with either a FACSCalibur or FACSCanto (Becton Dickinson, Franklin Lakes, NJ, USA) using Cellquest or DIVA (Becton Dickinson) software and were analysed using Flowjo software (Treestar, San Carlos, CA, USA). Viable cells were gated according to their forward and sideward scatter profiles and DAPI (Invitrogen) exclusion. CD4 and CD8 DN Thy1.2+ subsets were gated by lineage exclusion of CD4 and CD8 DP and SP cells and TCRγδ. DN subsets were further subdivided by CD25 and CD44 expression, defining CD25-CD44+ as DN1, CD25+CD44+ as DN2, CD25+CD44- as DN3 and CD25-CD44- as DN4, respectively. Cellular DNA content was measured by DAPI staining of methanol permeabilised cells. Phospho-S6 and phospho-Akt levels were assessed as described previously .
OP9 cultures and assay
OP9 bone marrow stromal cells expressing Notch ligand Delta Like-1 (DL-1) and control OP9 cells  were a gift from Juan Carlos Zúñiga-Pflücker (Toronto, Canada). OP9 cells were maintained in α MEM (Invitrogen) supplemented with 50 μM 2-mercaptoethanol, 100 U/ml penicillin, 1 mg/ml streptomycin and 20% heat-inactivated FBS. Sorted DN thymocytes were co-cultured on OP9 DL-1 or OP9 control cells for the indicated time periods. DN thymocytes were obtained using an AutoMACS (Miltenyi Biotech, Bergisch-Gladbach, Germany) magnetic cell sorter by depleting CD4+ and CD8+ thymic subpopulations to greater than 97% purity. DN3 or DN4 cells were obtained by subsequently either positively sorting or depleting CD25-expressing DN3 cells, respectively. Alternatively, DN3 cells were obtained by sorting with a FACSAria single cell sorter (Becton Dickinson, Heidelberg, Germany) with greater than 98% purity. On the day of harvest thymocytes were filtered through 50 μm filters to remove OP9 cells before assessing developmental progression and proliferation of T lineage cells.
Western blot analysis
Thymocytes or splenocytes were resuspended in lysis buffer containing 10 mMNaF, 1 mM sodium orthovanadate and protease inhibitor cocktail (Sigma) on ice for15 min. Lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes (Perkin-Elmer, Rodgau-Jügesheim). After blocking with 5% milk in TBST, membranes were probed with primary antibodies and subsequently detected using horseradish peroxidase-linked goat anti-mouse or anti-rabbit IgG and visualized by the enhanced chemilumescent (ECL) detection system (GE Healthcare, Munich, Germany). Membranes were reprobed with anti-β-actin (Sigma, St. Louis, Missouri, USA). The anti-SLY1 antibody has been generated by Eurogentec (Seraing, Belgium) and has already been described . Anti-phospho-SLY1 antibodies also have been generated by Eurogentec by immunizing rabbits with oligopeptide (H2N-LQR SSpS FKD FAK C-CONH2). Both antibodies have been tested extensively for specificity (data not shown).
Statistical significance of differences between wildtype and SLY1-targeted mice were evaluated using the Students t test. P < 0.05 was considered significant. All results are presented as mean values ± SEM.
We thank Prof. Doreen Cantrell and David Finlay for experimental advice and Prof. Juan Carlos Zúñiga-Pflücker who generously provided the OP9 DL-1 and OP9 GFP cells. We thank Nicole Küpper and Karin Buchholz for technical assistance.
This work was supported by grants BE 2813/1-1 (to S.B.) of the Deutsche Forschungsgemeinschaft (DFG), and the Forschungskommission of the Heinrich-Heine University Duesseldorf (to S.B.).
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