Title: PDK1 regulates VDJ recombination, cell-cycle exit and survival during B-cell development
Abstract: Article5 March 2013Open Access PDK1 regulates VDJ recombination, cell-cycle exit and survival during B-cell development Ram K C Venigalla Corresponding Author Ram K C Venigalla MRC Protein Phosphorylation Unit, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Victoria A McGuire Victoria A McGuire MRC Protein Phosphorylation Unit, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Rosemary Clarke Rosemary Clarke Division of Cell Signaling and Immunology, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Janet C Patterson-Kane Janet C Patterson-Kane Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK Search for more papers by this author Ayaz Najafov Ayaz Najafov MRC Protein Phosphorylation Unit, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Rachel Toth Rachel Toth MRC Protein Phosphorylation Unit, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Pierre C McCarthy Pierre C McCarthy MRC Protein Phosphorylation Unit, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Frederick Simeons Frederick Simeons Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Laste Stojanovski Laste Stojanovski Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author J Simon C Arthur Corresponding Author J Simon C Arthur MRC Protein Phosphorylation Unit, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, UK Division of Cell Signaling and Immunology, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Ram K C Venigalla Corresponding Author Ram K C Venigalla MRC Protein Phosphorylation Unit, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Victoria A McGuire Victoria A McGuire MRC Protein Phosphorylation Unit, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Rosemary Clarke Rosemary Clarke Division of Cell Signaling and Immunology, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Janet C Patterson-Kane Janet C Patterson-Kane Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK Search for more papers by this author Ayaz Najafov Ayaz Najafov MRC Protein Phosphorylation Unit, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Rachel Toth Rachel Toth MRC Protein Phosphorylation Unit, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Pierre C McCarthy Pierre C McCarthy MRC Protein Phosphorylation Unit, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Frederick Simeons Frederick Simeons Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Laste Stojanovski Laste Stojanovski Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author J Simon C Arthur Corresponding Author J Simon C Arthur MRC Protein Phosphorylation Unit, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, UK Division of Cell Signaling and Immunology, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Author Information Ram K C Venigalla 1, Victoria A McGuire1,‡, Rosemary Clarke2,‡, Janet C Patterson-Kane3, Ayaz Najafov1, Rachel Toth1, Pierre C McCarthy1, Frederick Simeons4, Laste Stojanovski4 and J Simon C Arthur 1,2 1MRC Protein Phosphorylation Unit, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, UK 2Division of Cell Signaling and Immunology, College of Life Sciences, University of Dundee, Dundee, UK 3Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK 4Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, UK ‡These authors contributed equally to this work. *Corresponding authors. Lymphocyte Signalling and Development, The Babraham Institute, Cambridge, CB22 3AQ, UK. Tel.:+44 1223 496467; E-mail: [email protected] of Cell Signaling and Immunology, College of Life Sciences, University of Dundee, Dundee, UK. Tel.:+44 1382 384003; Fax:+44 1382 385783; E-mail: [email protected] The EMBO Journal (2013)32:1008-1022https://doi.org/10.1038/emboj.2013.40 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Phosphoinositide-dependent kinase-1 (PDK1) controls the activation of a subset of AGC kinases. Using a conditional knockout of PDK1 in haematopoietic cells, we demonstrate that PDK1 is essential for B cell development. B-cell progenitors lacking PDK1 arrested at the transition of pro-B to pre-B cells, due to a cell autonomous defect. Loss of PDK1 decreased the expression of the IgH chain in pro-B cells due to impaired recombination of the IgH distal variable segments, a process coordinated by the transcription factor Pax5. The expression of Pax5 in pre-B cells was decreased in PDK1 knockouts, which correlated with reduced expression of the Pax5 target genes IRF4, IRF8 and Aiolos. As a result, Ccnd3 is upregulated in PDK1 knockout pre-B cells and they have an impaired ability to undergo cell-cycle arrest, a necessary event for Ig light chain rearrangement. Instead, these cells underwent apoptosis that correlated with diminished expression of the pro-survival gene Bcl2A1. Reintroduction of both Pax5 and Bcl2A1 together into PDK1 knockout pro-B cells restored their ability to differentiate in vitro into mature B cells. Introduction B cells play critical roles in the adaptive immune response and the formation of immunological memory. However if their development is not correctly regulated, B cells can give rise to serious pathologies. For instance, if self-reactive B cells are not deleted or repressed then they will lead to the development of autoimmunity, while mutations that activate B-cell proliferation can result in B-cell lymphomas or leukaemia (Nemazee, 2006; Herzog and Jumaa, 2012). It is therefore essential that B-cell development and proliferation are closely regulated and complex mechanisms have evolved to achieve this in vivo. How kinase signalling cascades contribute to this process is still not fully understood. AGC kinases represent a family of closely related enzymes including PKC, Akt, PKA and RSK, and members of this family have been implicated in cellular proliferation, survival and differentiation. The activation of AGC kinases involves the phosphorylation of a specific residue in their activation (or 'T') loop as well as the phosphorylation of a second site in the C-terminus, referred to as the hydrophobic motif. Phosphoinositide-dependent kinase-1 (PDK1) is required for the phosphorylation of the activation loop of a subset of AGC kinases, including Akt, S6K, RSK and PKCs (Downward, 1998; Vanhaesebroeck and Alessi, 2000; Mora et al, 2004; Pearce et al, 2010). T loop phosphorylation is essential for the activation of Akt, S6K and RSK and consequently these kinases are inactive in cells lacking PDK1 (Williams et al, 2000). PDK1 therefore serves as a master regulator of a subgroup of AGC kinases. PDK1 is constitutively active in cells, and its ability to phosphorylate its substrates is regulated at the level of protein interaction. PDK1 regulates its targets by one of two mechanisms. For Akt, both Akt and PDK1 are recruited to the membrane by binding of their respective PH domains to PIP3. This then permits PDK1 to phosphorylate the T loop (Thr308) of Akt, while the hydrophobic motif (Ser473) is phosphorylated by the mTORC2 complex (McManus et al, 2004; Pearce et al, 2010). Thus, the activation of Akt by PDK1 is dependent on PI-3 kinase activation, which catalyses the production of PIP3 at the membrane. For other PDK1 substrates, the phosphorylation of the hydrophobic motif at the C-terminus of the AGC kinase domain is required to generate a docking site for PDK1, and significantly for these substrates PDK1 can act independently of PI-3 kinase signalling (Collins et al, 2003). Once docked, PDK1 then phosphorylates the T loop residue of the downstream kinase. The hydrophobic motif is phosphorylated by a distinct kinase, for instance mTORC1 for S6K and either autophosphorylation or MK2 for RSK (Zaru et al, 2007; Pearce et al, 2010). Given the key role that PDK1 plays in signalling networks, it is an important kinase for several developmental processes. Knockout of PDK1 in mice results in embryonic lethality at E9.5 (Lawlor et al, 2002). Conditional knockout of PDK1 in muscle results in postnatal lethality, with the mice dying within 5–11 weeks of birth due to cardiac defects, while mice lacking PDK1 in the liver die within 4–16 weeks from liver failure (Mora et al, 2003, 2004). Conditional deletion in osteoclasts gives rise to skeletal defects that have a similarity to Rubinstein-Taybi syndrome (Shim et al, 2011). The role of PDK1 in B-cell development however has not been directly addressed. Recent studies have started to dissect the roles of the PI-3 kinase signalling pathway in lymphocyte development (Okkenhaug and Vanhaesebroeck, 2003; Lazorchak et al, 2010; Ramadani et al, 2010; Baracho et al, 2011). For instance, mice with a conditional deletion of PI3Kα and an inactivating mutation in PI3Kδ in B cells exhibit a developmental block at the pro to pre-B stage (Ramadani et al, 2010). This could imply a role for PDK1 in B-cell development, however, PI-3 kinase signalling can also result in GEF and Tec kinase activation independently of PDK1 and Akt (Fruman, 2004; Wertheimer et al, 2012). Thus, it is also possible that the effects of PI-3 kinase inhibition in B-cell development occur via these Akt-independent pathways. PDK1 has been shown to be important for the development of T cells (Hinton et al, 2004; Lee et al, 2005), loss of it results in T-cell developmental arrest at the DN3/4 stage in the thymus (Hinton et al, 2004). Deletion of PDK1 at the DN3 stage of development does not prevent recombination at the TCRβ locus, but does inhibit the progression of DN cells to the DP stage of development, resulting in very few T cells in the periphery (Hinton et al, 2004). T-cell development has also been shown to be sensitive to PDK1 levels; experiments using bone marrow from mice with a hypomorphic allele that results in ∼10% of the normal levels of PDK1 fail to competitively repopulate the T-cell pool in lethally irradiated mice. Interestingly in the same experiments, PDK1 had much less effect on B-cell repopulation (Hinton et al, 2004). In line with this, while knockout of Akt1 and 2 greatly perturbs T-cell development (Juntilla et al, 2007; Mao et al, 2007), it does not block B-cell development in the bone marrow (Calamito et al, 2009). The effect of Akt1/2 knockout in B cells was less severe that seen in the PI-3 kinase mutants (Ramadani et al, 2010). This difference may derive from several reasons, for example an Akt-independent function of PI-3 kinase in B-cell development or an upregulation of Akt3 activity in the Akt1/2 knockouts could explain these results. A further possibility is that the Akt knockout study used fetal liver cells to generate B cells, and it is possible that Akt may have less importance in embryonic rather than adult B cells. This suggests that PDK1 could be less critical for B-cell development; however, its role in this process has not been uncovered. To address this issue, we generated a conditional PDK1 knockout in the haematopoietic system using a Vav-Cre transgene. Interestingly, we observed that loss of PDK1 results in the arrest of B-cell development at the transition from pro-B to pre-B cells in the bone marrow. This was due to reduced Ig chain recombination that correlated with decreased expression of Pax5 and DNA ligase IV, proteins required for recombination events. In addition, PDK1 knockout pre-B cells underwent apoptosis due to a decrease in expression of the pro-survival gene Bcl2A1. The importance of Pax-5 and Bcl2A1 downstream of PDK1 was confirmed by the finding that reintroduction of both Pax5 and Bcl2A1 together into PDK1 knockout pre-B cells in vitro was required to restore their ability to differentiate into mature (IgD+ve) B cells. Results Loss of PDK1 in haematopoietic cells blocks T and B cells but not myeloid cell development To generate mice lacking PDK1 in haematopoietic cells, PDK1fl/fl mice were crossed to Vav-Cre transgenic mice, which express Cre early in haematopoietic development. Deletion of PDK1 was confirmed by qPCR of bone marrow, splenocytes and thymocytes. PDK1fl/fl/Vav-Cre+ve were smaller than littermate controls (Supplementary Figure 1) and showed evidence of increased myeloid cell recruitment into the lung and liver (Supplementary Figure 2). In the lung, this was noted around and within arterial and venous walls, and there was significant associated arterial muscular hypertrophy. Despite the decreased body size, 6- to 24-week-old PDK1fl/fl/Vav-Cre+ve mice had larger spleens relative to control genotypes (Figure 1A and B). However, while there was an increase in spleen size, following red blood cell lysis the splenocyte cell number was comparable between PDK1fl/fl/Vav-Cre+ve knockout mice and control animals (Figure 1C). H&E staining revealed that the white pulp in PDK1fl/fl/Vav-Cre+ve spleens was replaced by immature myeloid cells with increased numbers of granulocytes at various stages of maturity at the margins of this peri-arterial and peri-arteriolar tissue and throughout the red pulp. Increased numbers of siderophages were also noted. These observations indicated a defect in lymphocyte recruitment or development (Figure 1D). Consistent with the HE staining, FACS analysis of the splenocytes demonstrated that the PDK1-deficient spleens had an increased number of granulocytes and macrophages (Supplementary Figure 3). Normal numbers of conventional dendritic cells were found although the numbers of plasmacytoid dendritic cells was greatly reduced (Supplementary Figure 3). FACS analysis for TCRβ or B220-positive cells demonstrated that there were no clear mature B- or T-cell populations in the spleens of PDK1fl/fl/Vav-Cre+ve mice (Figure 1F and E), in agreement with the absence of a defined white pulp (Figure 1D). This lack of T and B cells was not restricted to the spleen, as lymph nodes in the PDK1 knockout mice were small and contained no mature lymphocytes (Supplementary Figure 4). The lack of lymphocytes in the secondary immune organs could be explained by either a failure in development or migration. Analysis of the blood of PDK1fl/fl/Vav-Cre+ve mice showed that there were no mature T or B cells present (Supplementary Figure 5), indicating that PDK1 was essential for either the development of T and B cells or their emigration from the lymphogenic organs. Deletion of PDK1 in the thymus at the DN3/4 stage of T-cell development has been shown to block T-cell development due to a decreased proliferation of DN4 cells and failure to upregulate CD4 and CD8 (Hinton et al, 2004). Deletion in the PDK1fl/fl/VavCre+ve mice occurs in the bone marrow, earlier than the Lck-Cre used by Hinton et al (2004). Analysis of the thymi from PDK1fl/fl/VavCre+ve mice demonstrated that there was an absence of CD4/CD8 DP cells and failure to upregulate the cell surface expression of TCRβ (Supplementary Figure 6). Development was arrested at the DN3 stage, however, expression of the intracellular TCRβ chain in DN3 cells was similar to that seen in wild-type cells (Supplementary Figure 6). Thus, PDK1 is essential for T-cell development, but not for recombination of the TCRβ locus. In T cells, PDK1 deletion has been correlated to decreased levels of the CD98 amino acid transporter and the transferrin receptor CD71, potentially resulting in metabolic stress as the DN4 cells proliferate (Kelly et al, 2007). In contrast, in B cells PDK1 knockout caused an increase in CD98 and CD71 levels in pro- and pre-B cells (Supplementary Figure 6), indicating that the roles of PDK1 may vary between T and B cells. Figure 1.PDK1 knockout in the haematopoietic system blocks the development of mature T and B cells. PDK1fl/fl/Vav-Cre+ve mice were found to have an increased spleen size (A) and weight (B) relative to PDK1+/+/Vav-Cre+ve control mice. Post lysis of red blood cells however spleen cell numbers were similar between the PDK1fl/fl/Vav-Cre+ve mice (n=15) and PDK1+/+/Vav-Cre+ve (n=18), PDK1+/+/Vav-Cre−ve (n=6) and PDK1fl/fl/Vav-Cre−ve (n=6) control genotypes (C). Error bars represent standard deviation. H&E staining of the spleens showed that PDK1 knockout resulted in a disruption of the white pulp (D). Analysis of T- and B-cell populations in the spleen by FACS (E, F) demonstrated that PDK1fl/fl/Vav-Cre+ve mice (n=7) had negligible numbers of mature T and B cells relative to PDK1+/+/Vav-Cre+ve (n=6), PDK1+/+/Vav-Cre−ve (n=6) and PDK1fl/fl/Vav-Cre−ve (n=4) control genotypes. Error bars represent the standard deviation of 4–7 mice per genotype. In (B, C and E) a P-value (Student's t-test) of <0.01 is indicated by **. Differences in (C) were not significant (t-test P>0.05). Download figure Download PowerPoint As the role of PDK1 in B-cell development has not been established, the reason for the lack of mature B cells was investigated further. To determine if this was cell extrinsic or intrinsic, reconstitution experiments were carried out in sublethally irradiated Rag2 knockout mice. Injection of wild-type bone marrow that had been depleted of T and B cells into Rag2 mice reconstituted both T- and B-cell populations. In contrast, bone marrow from PDK1fl/fl/Vav-Cre+ve mice was unable to effectively reconstitute either T or B cells in the Rag2 knockout mice (Supplementary Figure 7). To further examine this, we carried out competitive repopulation experiments. When a mixture of wild-type cells expressing either CD45.1 or CD45.2 markers were injected, both were able to give rise to mature B cells. However when a mixture of wild-type cells expressing CD45.1 and PDK1fl/fl/Vav-Cre+ve expressing CD45.2 were injected, only the wild-type cells were found to give rise to B cells in either the blood or spleen (Figure 2). Figure 2.The effect of PDK1 on B-cell development is cell intrinsic. Bone marrow was isolated from mice of the indicated genotypes and depleted of B220+ve and CD3+ve cells as described in Materials and methods. In all, 1:1 mixes of the depleted bone marrow from PDK1+/+/Vav-Cre−ve mice expressing the CD45.1 marker were mixed with either PDK1+/+/Vav-Cre+ve or PDK1fl/fl/Vav-Cre+ve cells expressing CD45.2. Cells were then injected into sublethally irradiated Rag2 knockout mice (A). The B-cell compartment in the blood (B) and spleen (C) is shown. Download figure Download PowerPoint PDK1 is required for the transition of pro-B to pre-B cells B cells develop in the bone marrow from CLPs (common lymphoid progenitors) and pass through a series of stages from pre-progenitor B (pre-pro-B) cells to progenitor B (pro-B) cells, precursor (pre-B) B cells and finally immature B cells which exit the bone marrow to mature in the periphery (Hardy and Hayakawa, 2001; Bartholdy and Matthias, 2004; Fuxa and Skok, 2007; Monroe and Dorshkind, 2007). As the PDK1fl/fl/Vav-Cre+ve mice had no peripheral B cells, we examined B-cell development in the bone marrow. Total bone marrow cell numbers in the PDK1fl/fl/Vav-Cre+ve mice were comparable to control genotypes (Figure 3A). FACS analysis for HSCs showed that they were present at the expected frequencies in the PDK1 knockout. CMP (common myeloid progenitors) and CLP populations were also present but there was an increased frequency of CMPs in PDK1fl/fl/Vav-Cre+ve bone marrow relative to wild type (Figure 3B). CLPs may also be increased, although the resolution of this population in the knockouts was complicated by an increased c-kit+ve/Sca-1low cell population in this genotype. There was a trend for a lower number of total B-cell progenitors in the knockout bone marrow, however, this did not reach statistical significance (P>0.05, Student's t-test). Normal numbers of pre-pro-B cells were present in PDK1fl/fl/Vav-Cre+ve mice; however, there was a greatly increased number of pro-B cells relative to control mice and significantly fewer pre-B cells. No immature or mature B cells were found in the bone marrow of PDK1fl/fl/Vav-Cre+ve mice (Figure 3C and D). An alternative staining protocol that also distinguishes pro-B (B220+veIgM−vec-Kit+veCD25−ve) and pre-B cells (B220+veIgM−vec-kit−veCD25+ve) (Xiao et al, 2007) also gave similar results (Supplementary Figure 8A). Loss of PDK1 therefore results in a block in B-cell development primarily at the pro- to pre-B cell transition. This effect was cell intrinsic as competitive repopulation experiments in Rag2 knockout mice demonstrated that cells lacking PDK1 were unable to reconstitute the pre-B cell population in the bone marrow (Figure 3E). Figure 3.Loss of PDK1 blocks B-cell development at the pro-B cell developmental stage. Total bone marrow cell numbers were similar between PDK1fl/fl/Vav-Cre+ve and control genotypes (A). Error bars represent the standard deviation of 6–16 mice per genotype. Differences were not significant (t-test, P>0.05). Cell development in the bone marrow was further examined by FACS. Staining of Lin−veCD127+ve cells with c-Kit and Sca1 was used to identify CLPs while staining of the Lin−veCD127−ve cells identified the MLP (c-kit+ve/Sca1−ve) and HSC (c-kit+ve/Sca1+ve) compartments. Results are representative of FACS on three mice per genotype (B). Staining of B220+ve cells for IgM demonstrated that loss of PDK1 blocked the development of immature (B220low/IgM+ve) and mature (B220hi/IgM+ve) cells (C, D). CD19/CD43 staining of the B220+ve/IgM−ve cells showed that PDK1 knockout resulted in an accumulation of pro-B cells but a drastic decrease in pre-B cell numbers (D, lower panels). Representative plots are shown in (D) and quantification of absolute cell numbers based on this shown in (C). Error bars in (C) represent the standard deviation of 4–5 mice per genotype. A P-value (Student's t-test) of <0.01 is indicated by ** and 0.05 by *. Competitive repopulation experiments (E) into Rag2 knockout mice demonstrated that while injected wild-type cells could give rise to immature B cells, PDK1fl/fl/Vav-Cre+ve cells were unable to differentiate into B220+veIgM+ve/CD43−ve immature B cells but were instead arrested as pro-B cells (B220+veIgM−ve/CD43+ve). Download figure Download PowerPoint PDK1 regulates Ig heavy chain rearrangement The transition from pro-B to pre-B cells requires the rearrangement of the IgH locus to allow expression of a pre-BCR (Hardy and Hayakawa, 2001; Bartholdy and Matthias, 2004). Analysis of the intracellular levels of IgH by FACS in pro-B cells demonstrated that fewer PDK1 knockout cells expressed high levels of heavy chain relative to control cells (Figure 4A). We therefore looked at the effect of PDK1 knockout on V-DJ recombination. V-DJ recombination was reduced in PDK1 knockout pro-B cells, an effect that was more apparent at distal (VHJ588) rather than proximal (VHJ7183) VH regions (Figure 4B and C). Heavy chain recombination requires the expression of the recombinase genes Rag1 and Rag2. Analysis of Rag1 and 2 mRNA expression showed that loss of PDK1 resulted in a two- to four-fold increase in the mRNA for these proteins (Figure 4D). Thus, failure to transcribe Rag genes would not account for the defect in V-DJ recombination. Although PDK1 knockout pro-B cells did not exhibit significantly different Pax5 mRNA (P<0.05) to wild-type cells (Figure 4D), Pax5 protein expression was slightly decreased in these cells (see Figure 7D). The mRNA levels of Artemis, Ku70, Ku80, DNA-PKc and XRCC4, which are also involved in recombination (Helmink and Sleckman, 2012; Schatz and Ji, 2011), were not significantly altered by PDK1 knockout, while levels of TdT mRNA were increased. By contrast, PDK1 knockout resulted in an ∼60% decrease in the expression of DNA Ligase IV (Figure 4D), an enzyme required for non-homologous end joining during IgH rearrangement (Frank et al, 1998; Grawunder et al, 1998). Figure 4.PDK1 deficiency impairs V-DJ recombination. Staining for the presence of intracellular heavy chain in CD19+veIgM−veCD43highB220+ve bone marrow (pro-B) cells demonstrated that while PDK1+/+Vav-Cre+ve cells were able to express the heavy chain, the levels of intracellular heavy chain were reduced in the PDK1fl/fl/Vav-Cre+ve cells. Results are representative of six mice (A). To examine V-DJ recombination, pro-B cells were isolated from PDK1+/+/Vav-Cre+ve and PDK1fl/fl/Vav-Cre+ve mice using FACS sorting. The heavy chain region was amplified by PCR and Southern blotting used to examine recombination for distal (VHJ588) and proximal (VHJ7183) rearrangements (B). V-DJ mRNA expression as well as the expression of the VHJ7183 and VHJ588 germline transcripts was also analysed using qPCR (C) with similar results. Total RNA was isolated from FACS-sorted pro-B cells and the levels of genes involved in V-DJ recombination were determined by qPCR (D). Expression levels were determined relative to HPRT and error bars represent the standard deviation of RNA preparations from FACS-sorted pro-B cells from three independent pools of mice per genotype. Loss of PDK1 resulted in significant increases in the mRNA levels for Rag1, Rag2 and TdT and decreased levels of DNA Ligase IV. In (C, D), a P<0.05 is indicated by * and P<0.01 by ** while NS indicates P>0.05 (Student's t-test). Download figure Download PowerPoint Loss of PDK1 inhibits Akt and RSK activation While PDK1 deficiency reduced recombination at the IgH locus, it was not totally blocked and therefore may not completely explain the absence of B cells. Once heavy chain is expressed, it combines with the VpreB, λ5, Igα and Igβ chains to form a functional pre-BCR (Milne et al, 2004; Herzog et al, 2009). mRNA expression of these pre-BCR components in the PDK1fl/fl/Vav-Cre+ve pro-B cells was similar to wild-type (Figure 5A). Analysis of the levels of intracellular heavy chain in the PDK1 knockout pre-B cell gate demonstrated that it was expressed in the majority of these cells (Figure 5B; Supplementary Figure 8). The increased expression of the intracellular heavy chain in the pre-B cell gate relative to pro-B cells (compare Figures 4A and 5B) is consistent with some PDK1 knockout cells being able to make the transition from pro-B to pre-B cells. The low numbers of these cells and their failure to develop further would however suggest that PDK1 plays important roles at the pre-B cell stage. In these cells, PDK1 could act downstream of several signals, including IL-7 and the pre-BCR. To confirm that the knockout of PDK1 in developing B cells was sufficient to block the activation of its downstream targets, pro-B cells were expanded in IL-7 and then after IL-7 withdrawal stimulated with anti-Igβ and the activation of intracellular signalling pathways analysed by immunoblotting (Figure 5C). Both wild-type and PDK1fl/fl/Vav-Cre+ve cells were able to activate ERK1/2, indicating that both genotypes could respond to the anti-Igβ stimulation. RSK is activated by a complex mechanism involving phosphorylation by both ERK1/2 and PDK1 (Pearce et al, 2010). While ERK1/2 was activated normally in the PDK1fl/fl/Vav-Cre+ve cells the phosphorylation of RSK on the PDK1 site, S227, was inhibited. As S227 is critical for RSK activity (Pearce et al, 2010), this indicates that RSK would be inactive in these cells. PDK1 is also required for the activation of p70S6K (Pearce et al, 2010), and consistent with this the PDK1fl/fl/Vav-Cre+ve cells had no phosphorylation of the p70S6K substrate S6 (Figure 5C). Figure 5.PDK1 is required for Akt, p70S6K and RSK2 activation in B cells. (A) The mRNA levels for the pre-BCR components λ5, Igα, Igβ and VpreB were determined by qPCR from total RNA isolated from FACS-sorted pro-B cells from either PDK1+/+Vav-Cre+ve or PDK1fl/flVav-Cre+ve mice. Error bars represent the standard deviation of RNA from three independent pools of mice per genotype. (B) The levels of intracellular heavy chain were determined by FACS staining of ex vivo bone marrow cells, gating on the CD19+veIgM−veCD43−veB220+ve cells to examine pre-B cells. (C) Pro-B cells were isolated from PDK1+/+Vav-Cre+ve and PDK1fl/flVav-Cre+ve mice and stimulated for 6 days with 10 ng/ml IL-7. After withdrawal of IL-7 for 4 h, cel