PDK1 regulates B cell differentiation and homeostasis

Gisele Baracho

Matthew Cato

Zilu Zhu

Olav Jaren

Elias Hobeika

Michael Reth

Robert Rickert

Supplementary Material

Supporting Information:

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^{Program on Immunity and Pathogenesis, Sanford–Burnham Medical Research Institute, La Jolla, CA, 92037; and}

^{BIOSS Centre for Biological Signalling Studies and Department of Molecular Immunology, Bio III, Faculty of Biology, University of Freiburg and Max Planck Institute for Immunobiology and Epigenetics, 79108 Freiburg, Germany}

^{To whom correspondence should be addressed. E-mail:}gro.mahnrubdrofnas@trebor.

Edited by John C. Cambier, University of Colorado School of Medicine, Denver, CO, and accepted by the Editorial Board May 16, 2014 (received for review August 1, 2013)

Author contributions: G.V.B., M.H.C., O.R.J., and R.C.R. designed research; G.V.B., M.H.C., Z.Z., and O.R.J. performed research; E.H. and M.R. contributed new reagents/analytic tools; G.V.B., M.H.C., O.R.J., and R.C.R. analyzed data; and G.V.B. and R.C.R. wrote the paper.

Edited by John C. Cambier, University of Colorado School of Medicine, Denver, CO, and accepted by the Editorial Board May 16, 2014 (received for review August 1, 2013)

Significance

Phosphatidyl inositol-3 kinase (PI3K) activity is central to B lymphocyte survival, growth, and differentiation. Recent clinical studies also indicate that inhibiting the activity of the PI3Kδ isoform will be effective in treating human B cell malignancies. Here we focus on the B cell-specific role of the Ser/Thr kinase phosphoinositide-dependent kinase 1 (PDK1), a pivotal downstream effector of PI3K signaling that is required for the activation of most members of the AGC kinase family (e.g., Akt, S6K, RSK, PKC). Using conditional gene-targeting approaches, we show that the B cell receptor and a subset of cytokine receptors require PDK1 for B cell generation in the bone marrow as well as for mature B cell survival and activation.

Significance

Abstract

Successful B cell differentiation and prevention of cell transformation depends on balanced and fine-tuned activation of cellular signaling pathways. The phosphatidyl inositol-3 kinase (PI3K) signaling pathway has emerged as a major regulator of B lymphocyte homeostasis and function. Phosphoinositide-dependent protein kinase-1 (PDK1) is the pivotal node in the PI3K pathway, regulating the stability and activity of downstream AGC kinases (including Akt, RSK, S6K, SGK, and PKC). Although the importance of PI3K activity in B cell differentiation is well documented, the role of PDK1 and other downstream effectors is underexplored. Here we used inducible and stage-specific gene targeting approaches to elucidate the role of PDK1 in early and peripheral B cell differentiation. PDK1 ablation enhanced cell cycle entry and apoptosis of IL-7–dependent pro-B cells, blocking Ig synthesis and B cell maturation. PDK1 also was essential for the survival and activation of peripheral B cells via regulation of PKC and Akt-dependent downstream effectors, such as GSK3α/β and Foxo1. We found that PDK1 deletion strongly impaired B cell receptor (BCR) signaling, but IL-4 costimulation was sufficient to restore BCR-induced proliferation. IL-4 also normalized PKCβ activation and hexokinase II expression in BCR-stimulated cells, suggesting that this signaling pathway can act independent of PDK1 to support B cell growth. In summary, our results demonstrate that PDK1 is indispensable for B cell survival, proliferation, and growth regulation.

Abstract

Activation of the phosphatidyl inositol-3 kinase (PI3K) signaling pathway is critical to early B cell development as well as peripheral B cell survival and activation (1). Although the catalytic p110 subunits of class I PI3K molecules are partially redundant, the combined loss of the p110α and p110δ isoforms results in impaired IL-7R–driven proliferation (2). Conversely, it has been suggested that attenuation of PI3K signaling via IL-7R signaling is required for pre-B cell differentiation into IgM-expressing cells to cease proliferation and promote RAG expression (3).

In peripheral B cells, continued survival requires “tonic” signaling via the B cell receptor (BCR), which can be replaced by constitutive PI3K activity (4). Moreover, generation of the marginal zone (MZ) and B-1 B cell subsets, as well as antigen-driven differentiation into antibody-producing cells, are dependent on PI3K (1). PI3K activity generates PtdIns(3,4,5)P₃, which acts as a secondary messenger by binding the pleckstrin homology domains of downstream effector molecules. PtdIns(3,4,5)P₃ is also the substrate for the phosphatases PTEN and SHIP, generating PtdIns(4,5)P₂ and PtdIns(3,4)P₂, respectively. Unrestrained activation of PI3K signaling in B cells lacking PTEN and SHIP results in lethal B cell lymphoma (5).

Phosphoinositide-dependent kinase 1 (PDK1) represents a pivotal downstream effector of PI3K signaling, regulating cellular responses to growth factors, insulin, and numerous other agonists by activating a number of AGC protein kinases. Analysis of Pdk1^{mouse embryonic stem cells confirmed that the major targets include protein kinase B (PKB)/Akt, p70 and p90 ribosomal S6 kinases (S6K1 and RSK, respectively), serum-and glucocorticoid-induced protein kinase (SGK), and protein kinase C (PKC) isoforms (}6). After cell stimulation, phosphorylation of conserved residues in the carboxyl-terminal domain of the AGC kinases creates a docking site for PDK1 (7). Maximum activity and stability of the AGC kinases depend on the subsequent phosphorylation of a key threonine residue in the T or activation loop within the catalytic domain by PDK1 (7). The mechanism of Akt activation is an exception to this general rule. Akt isoforms as well as PDK1 have a pleckstrin homology domain that specifically binds to PtdIns(3,4,5)P₃. This interaction induces translocation and colocalization of Akt and PDK1 to the inner membrane, where PDK1 can effectively phosphorylate the T loop of Akt at T308 (8). The Sin1/Rictor-containing mTOR complex (mTORC2) phosphorylates Akt at S473 in the hydrophobic C-terminal motif (9), resulting in dual phosphorylation and full activation. In the present study, we used a genetic approach to explore the functions of PDK1 in B cell development and differentiation.

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Acknowledgments

We thank Drs. K. Rajewsky and M. Shlomchik for providing the Cd21^andhCD20Tam^{mice, respectively, and the Rickert laboratory for discussions. This work was supported by National Institutes of Health Grants AI041649 and}{"type":"entrez-nucleotide","attrs":{"text":"AI059447","term_id":"3333224","term_text":"AI059447"}}AI059447 (to R.C.R.) and German Science Foundation Grants TRR130 and SFB746 (to M.R.).

Acknowledgments

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. J.C.C. is a guest editor invited by the Editorial Board.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1314562111/-/DCSupplemental.

Footnotes

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