PDK1 regulates VDJ recombination, cell-cycle exit and survival during B-cell development.

Ram K C Venigalla,Laste Stojanovski,Janet C Patterson-Kane,Rachel Toth,Frederick Simeons,Ayaz Najafov,Victoria A Mcguire,J Simon C Arthur,Pierre C Mccarthy,Rosemary Clarke

doi:10.1038/emboj.2013.40

Abstract

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.

Highlights

B cells play critical roles in the adaptive immune response and the formation of immunological memory
FACS analysis for TCRb or B220-positive cells demonstrated that there were no clear mature B- or T-cell populations in the spleens of PDK1fl/fl/Vav-Cre þ 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 Phosphoinositide-dependent kinase-1 (PDK1) knockout mice were small and contained no mature lymphocytes (Supplementary Figure 4)
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

Summary

Introduction

B cells play critical roles in the adaptive immune response and the formation of immunological memory. 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 these kinases are inactive in cells lacking PDK1 (Williams et al, 2000). For Akt, both Akt and PDK1 are recruited to the membrane by binding of their respective PH domains to PIP3 This 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). 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)

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Results

Conclusion